CN114072414A

CN114072414A - Coumarin-modified androgens for the treatment of prostate cancer

Info

Publication number: CN114072414A
Application number: CN202080046675.3A
Authority: CN
Inventors: J·L·默勒; M·V·芬达洛; D·沃特; V·斯维瑞帕
Original assignee: Health Research Inc; University of Kentucky Research Foundation
Current assignee: Health Research Inc; University of Kentucky Research Foundation
Priority date: 2019-04-27
Filing date: 2020-04-27
Publication date: 2022-02-18
Also published as: WO2020223174A1; EP3962921A4; EP3962921A1; US20220218722A1; CA3138176A1; JP2022529745A

Abstract

The present invention provides androstane and dihydrotestosterone compounds functionalized with a carbocyclic or heterocyclic group which may be saturated or unsaturated. The compounds are useful in methods of inhibiting cell growth of malignant and/or proliferating cells and/or treating individuals having a disease associated with malignant cell growth (e.g., cancer, such as prostate cancer) and/or proliferating cell growth and/or imaging malignant and/or proliferating cell molecules and/or inducing degradation of a target protein. The invention also provides compositions.

Description

Coumarin-modified androgens for the treatment of prostate cancer

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims U.S. provisional application No. 62/839,676, filed 2019, month 4, 27; us provisional application No. 62/844,062 filed on 6.5.2019; us provisional application No. 62/844,073 filed on 6.5.2019; and us provisional application No. 62/890,292 filed 2019, 8, 22, the disclosure of which is incorporated herein.

Statement regarding federally sponsored research

The invention was made with government support under contract numbers CA77739, P20 RR020171, R21 CA2051 and P30 GM110787 awarded by the national cancer institute and contract numbers W81XWH-16-1-0635 and W81XWH-15-1-0409 awarded by the department of defense. The government has certain rights in the invention.

Background

Growth and progression of prostate cancer (Cap) is dependent on Androgen Receptor (AR) and circulationThe interaction between androgens (testosterone (T) produced by the testes and its metabolites, 5 α -Dihydrotestosterone (DHT) produced by the 5 α reduction of T in prostate tissue). T and DHT have similar affinity for AR at the Ligand Binding Domain (LBD), but DHT is more potent because its off-rate is slower. Ligand-bound AR dimerizes and translocates to the nucleus, binds to Androgen Response Elements (ARE) and initiates transcription of AR regulatory genes that promote growth of CaP cells. Most men with metastatic disease or those who may fail curative therapy receive Androgen Deprivation Therapy (ADT) treatment which reduces circulating T levels, impairs AR transactivation and leads to CaP suppression. However, ADT is palliative, and CaP invariably relapses in the form of lethal castration-relapsing/resistant CaP (crpc). One mechanism that contributes to the shift of CaP to CRPC is intratumoral androgen metabolism. In the absence of circulating T during ADT, androgens, dehydroepiandrosterone, or 4-androstene-3, 17-dione (ASD) are converted to DHT by T via the front door (frontdoor) pathway (fig. 1). CaP cells use two other pathways to synthesize DHT that do not require T as an intermediate. In the main backdoor (back door) pathway, 5 α -androstan-3 α -ol-17-one is enzymatically reduced to 5 α -androstan-3 α,17 β -diol (diol), which in turn is enzymatically oxidized to DHT (fig. 1). In the minor backportal pathway, ASD is enzymatically reduced to 5 α -androstane-3, 17-dione (5 α -dione), which in turn is enzymatically reduced to DHT (fig. 1). In summary, three enzymatically driven pathways provide a means of contacting DHT to CaP tissues, and each pathway has enzymes that catalyze key steps late in the biosynthetic pathway. Finasteride or dutasteride inhibits the 5 α -reductase (SRD5A) to reduce inhibition of T to DHT conversion (i.e., the front gate pathway in fig. 1). Both drugs fail clinically, in part because the backdoor pathway produces sufficient DHT levels to activate AR and CaP growth. Still other inhibitors, such as the cytochrome P45017A 1(CYP17A1) inhibitor abiraterone, inhibit pregnane (C45017A 1) before the terminal parts of the three pathways converge on DHT (C1)₂₁Compound) to androstane (C)₁₉Compound) is synthesized. Abiraterone (and other CYP17a1 inhibitors) only prolonged survival in CRPC for several months, as several mechanisms allow for enhanced DHT production and CaP growth, a mechanism that includes elevated CYP17a1 expression levels and accumulation of progesterone (which may exceed abiraterone for CYP17A, 25CYP11a1 or AKR1C 3).

ASD is metabolized to DHT via the anterior and 2 posterior pathways, using the same metabolic steps in a different order at C-3 and C-17 (FIG. 1). ASD either goes through T to DHT or into 5 α -diketones, which go either directly into DHT or through androsterone, then diol, and finally DHT (fig. 1). The secondary backportal pathway is named after the primary backportal pathway it found, synonymous with the "alternative" by Sharifi, the "alternative" by Penning and the "5 α -diketone" pathway by Corcoran. The role of HSD17B3 in the metabolism within the tumor via the anterior and secondary posterior pathways can be isolated using ASD and 5 α -diketone, respectively, as substrates. A preclinical study with the AKR1C3 inhibitor indomethacin demonstrated that inhibition of AKR1C3 overcomes CaP resistance to abiraterone and enzalutamide. This study provided evidence for the necessity to identify and develop AKR1C3 inhibitors. A phase 1/2 clinical trial testing the efficacy of the AKR1C3 inhibitor ASP9521 ended without evidence of clinical response. The authors concluded that the insufficient CaP cell expression of AKR1C3 resulted in treatment failure and that the four 3 α -oxidoreductases discussed herein may not present this problem.

In methods of achieving ADT, antiandrogens occupy a central position in chemically castrating men with CaP. The antiandrogen bicalutamide or enzalutamide competes with T or DHT for AR-LBD. Since bicalutamide has a lower binding affinity for AR-LBD than DHT, it does not perform well clinically. In addition, bicalutamide exhibits undesirable AR agonist activity after long-term treatment rather than antagonist activity. Enzalutamide binds with higher affinity to AR-LBD than bicalutamide, impairs AR nuclear translocation, and inhibits the interaction between ARE and AR of AR regulatory genes critical to CaP growth. However, enzalutamide produced a modest response in CRPC and extended survival by only 4.8 months. One mechanism that leads to abiraterone and antiandrogen resistance is the expression of the AR splice variant 7(AR-V7), which is constitutively active and lacks LBD. The lack of LBD obviates the need for T or DHT and eliminates the binding site for antiandrogens, rendering them ineffective. In vitro studies, small molecule inhibitors that target the N-terminal domain of AR, but not AR-LBD (e.g., EPI-506), inactivate AR-V7. EPI-506 was found to be safe in phase I trials, but failed phase 2 clinical trials. The limited success currently achieved with therapies directed to advanced CaP, and CRPC in particular, necessitates the pursuit of new therapies.

Disclosure of Invention

The present disclosure provides androstane and dihydrotestosterone compounds functionalized with carbocyclic or heterocyclic groups, which may be saturated or unsaturated (e.g., coumarin-containing ring groups (e.g., coumarin groups) and coumarin isostere groups). The compounds are useful in methods of inhibiting cell growth of malignant and/or proliferating cells and/or treating individuals having a disease associated with malignant cell growth (e.g., cancer, such as prostate cancer) and/or proliferating cell growth and/or imaging malignant and/or proliferating cell molecules and/or inducing degradation of a target protein.

The compound may be 1): inhibition of oxidoreductases that appear in the terminal steps of the major backgate pathway of DHT biosynthesis and/or 2) competition for AR-LBD binding and inhibition of cell growth in CaP cell lines expressing high levels of AR-V7 variants (possibly by impairing AR-V7 dimerization), thus acting as an antiandrogen and/or 3) providing a tool carrying small molecules that disrupt AR function and even degrade AR. Compounds with one or more of these properties would represent a significant clinical advance.

In one aspect, the present disclosure provides functionalized androstane and dihydrotestosterone compounds. The compounds can be modified with a coumarin ring group or a coumarin isostere group.

In various examples, the compounds of the present disclosure include 5 α -androstane-3 α,17 β -diol (diol), 5 α -androstane-3 α -ol-17-one, 5 α -androstane-3, 17-dione (5 α -dione), and 5 α -Dihydrotestosterone (DHT), which may be modified with various saturated or unsaturated heterocyclic or carbocyclic rings. In various examples, the compounds can be modified at the C-3 or C-17 position in these steroids with coumarin-containing groups or coumarin isostere groups.

The compounds of the present disclosure may have the following structure:

wherein R is¹Are hydrogen or alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, etc.). R³Selected from the group consisting of carbonyl,

Wherein X is hydroxyl and Y is hydrogen or an alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, etc.) or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or a coumarin isostere group, and L is³Is optional and is a linking group. R⁴Selected from the group consisting of carbonyl,

Wherein L is¹Is a linking group, L₂Is optional and is a linking group, Z is a terminal group comprising a substituted or unsubstituted carbocyclic group or a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted coumarin isostere group, and R is²Is an alkyl group (e.g., methyl group, ethyl group, n-propyl and isopropyl groups, etc.) or hydrogen. R⁵Is a photoactive group. When R is³Is a carbonyl group or

Wherein X is hydroxy and Y is hydrogen or an alkyl group, R⁴Is not provided with

Or a carbonyl group.

In one aspect, the present disclosure provides a composition comprising a compound of the present disclosure. The compositions also comprise one or more pharmaceutically acceptable carriers.

In one aspect, the present disclosure provides methods of using one or more compounds or compositions thereof. The compounds are suitable for use in methods of treating various diseases. For example, one or more compounds of the present disclosure or compositions of the present disclosure can be used to treat cancer, other diseases, or a combination thereof. The methods of the present disclosure are useful for inhibiting cell growth of malignant and/or proliferating cells and/or inducing selective degradation and/or molecular imaging of target proteins. A method may be performed in combination with one or more known therapies.

In one aspect, the present disclosure provides a kit or kit. In various examples, a kit or kit comprises a pharmaceutical formulation comprising any one or any combination of the compounds of the present disclosure. In one example, the present disclosure includes a closed or sealed package containing a pharmaceutical formulation. In various examples, the package includes one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for selling, distributing, or using the pharmaceutical compounds and compositions containing them. The printed material may include printed information. The printed information may be provided on a label, on a paper insert or printed on the packaging material. The printed information may include information identifying the compound in the package, the amount and type of other active and/or inactive ingredients in the composition, and instructions for administering the compound and/or composition. The instructions may include information such as the number of doses taken over a given period of time, and/or information directed to a pharmacist and/or other healthcare provider such as a doctor or patient. The printed material may include indications of the pharmaceutical composition and/or any other agents provided therein for treating a subject having prostate cancer (e.g., castration-resistant prostate cancer) and/or other diseases and/or any conditions associated with cancer and/or other diseases. In various examples, the kit or kit includes a label describing the kit or kit contents and providing indications and/or instructions regarding the use of the kit or kit contents to treat a subject having any cancer and/or other disease.

Brief description of the drawings

For a fuller understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken together with the accompanying figures.

FIG. 1 shows (A and B) biosynthetic pathways of DHT, including the anterior portal pathway, the major posterior portal pathway, and the minor posterior portal pathway.

FIG. 2 shows a synthetic route to fluorescent steroids modified at the C-17. beta. hydroxyl group. Description of reagents used in the synthesis: a, N₂CH₂CO₂Et，Rh₂(OAc)₄，CH₂Cl₂(ii) a b, NaOH, aqueous CH₃OH; c, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de ] hydrochloride]Anthracen-10-one; d, LiAlH (OtBu)₃；e，C₆H₅CO₂H，DIAD。

FIG. 3 shows a synthetic route to fluorescent steroids modified on the C-17 keto group. Description of reagents used in the synthesis: a, H₂NOCH₂CO₂Et; b, NaOH, aqueous CH₃OH; c, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de ] hydrochloride]Anthracen-10-one; d, Py-SO₃。

FIG. 4 shows a Pictet-Pengler (Pictet-Spengler) reaction of DHT with 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de ] anthracen-10-one hydrochloride. Description of reagents used in the synthesis: a, 1:10 (v/v) concentration of HCl-abs. Ethanol and refluxing.

FIG. 5 shows a 2D ROESY spectrum of DHT-3C (also known as Compound Ig). Spectra recorded at 25 ℃ using Agilent (Agilent) 400 MHz. The dotted line shows an enlarged area showing NH₂+ and ring A protons (i.e., H-5 (overlapping with H-1), H-4, H-2 and CH₂And NH₂+ adjacent) are connected by the Nuclear Overhauser Effect (NOE).

Fig. 6 shows the results of computational modeling. FIG. A: comprising a proximal NADP⁺The 5 α -diketone in the SP1 binding site of AKR1C3(PDB: 1XF 0). FIG. B: comprising a proximal NADP⁺Compound Ig in the SP1 binding site of AKR1C3(PDB: 1XF 0).

Fig. 7 shows the results of computational modeling. FIG. A: computational models of DHT-3C binding to AR indicate that the BCD loop of DHT-3C is inserted into the AR-LBD binding pocket. And B: an expanded view of DHT-3C binding shows interaction with specific residues in the AR and shows that the hairpin domain of the AR (759-771) is "opened" from its initial position by about

To accommodate DHT-3C.

FIG. 8 shows the expression of 3 α -oxidoreductase in clinical specimens.

FIG. 9 shows that RDH16 expression increases LAPC-4DHT production.

FIG. 10 shows ARE-luciferase signals generated by diol and DHT treatment. In each series, the columns from left to right are SFM, DHT, diol, AND Androsterone (AND).

Figure 11 shows that the combination of dutasteride and the 3 α -oxidoreductase mutant reduced DHT levels better than dutasteride alone. In each series, the columns from left to right are "empty vectors", HSD17B6, RDH16, DHRS9, and RDH 5.

FIG. 12 shows that RDH16 knockdown impairs DHT production in LAPC-4 cells.

Figure 13 shows that inhibition of DHT synthesis in the anterior and major posterior gates impairs VCaP growth.

Figure 14 shows that diol-17C treatment impaired VCaP cell growth.

FIG. 15 shows ImageStream RDH16-YFP and diol-17C co-localization.

FIG. 16 shows diol-17C and diol competing for RDH 16-YFP.

FIG. 17 shows that RDH16 metabolizes diol or diol-17C to DHT or DHT-17C, respectively.

FIG. 18 shows ImageStream data demonstrating a similar AR-GFP co-localization pattern between DHT-17C and DHT-C3. ImageStream also demonstrated nuclear co-localization between DHT-17C and AR-V2-GFP.

FIG. 19 shows competition data demonstrating that DHT-17C and enzalutamide compete for AR-V2-GFP.

FIG. 20 shows growth data demonstrating that DHT-17C impairs growth of VCaP and CWR-1 cell lines expressing AR and AR-V7, but not PC-3 (AR-free CaP cell lines) or non-CaP, non-AR CV-1 or 293 cell lines.

FIG. 21 shows growth data demonstrating that DHT-17C (20nM) inhibits AR-V7 positive VCaP growth at a dose 1000-fold lower than benoxalug (20 μ M).

FIG. 22 shows growth data demonstrating that the growth barrier between DHT-17C (50nM) and enzalutamide (20 μ M) is similar using the castration relapsed AR-V7 positive CRPC CWR-R1 cell line.

FIG. 23 shows qRT-PCR data demonstrating that DHT-17C treatment of CWR-R1 impairs transcription induction of the AR regulated gene kallikrein-associated peptidase 2(KLK 2).

FIG. 24 shows ImageStream data demonstrating nuclear co-localization between DHT-3C and AR-GFP (rather than free coumarin and AR-GFP). The AR-GFP study was confirmed using AR-V2-GFP.

FIG. 25 shows growth data demonstrating a similar growth pattern between AR positive CaP cell lines treated with DHT-3C or DHT. Each cell line is (A) LAPC-4; (B) VCaP; (C) LNCaP; (D) CRW-R1; (E)22rv 1; (F) PC-3; and (G) DU 145.

FIG. 26 shows that DHT-3C or DHT treatment of CaP cell lines results in similar expression levels of the AR regulatory gene KLK2 protein. Each cell line is (A) LAPC-4; (B) VCaP; (C) LNCaP; (D) CRW-R1; and (E)22rv 1.

Figure 27 shows a three-fold underinvestigation of AR ligand binding, AR dimerization, and AR transactivation.

FIG. 28 shows the utility of diol-17C (Compound Ia), DHT-17C (Compound Id), and DHT-3C (Compound Ig). diol-17C inhibits the intratumoral synthesis of DHT by oxidoreductase, DHT-17C competes with DHT for AR-LBD and may prevent dimerization between AR and AR-V7, and DHT-3C provides a vehicle for PROTACS or light or heat activators to disrupt AR transactivation and even degrade AR.

FIG. 29 shows representative photoactive derivatives of DHT-3C or C-3O- (carboxymethyl) oxime of DHT.

Fig. 30 shows a synthetic route to synthesize PROTAC for AR degradation.

Figure 31 shows the synthesis and pictet-spengler reaction of various 4- (2-aminoethyl) coumarins with DHT or 17 α -methyl-5 α -dihydrotestosterone.

FIG. 32 shows the effect of fluorescent 5 α -dihydrotestosterone replacement (DHT-C). Structure of DHT-C. Schematic representation of the transcriptional induction of genes that dht-C activates AR and stimulates AR regulation.

FIG. 33 shows a synthetic route to diol-17C and several analogs. (A) Synthesizing diol-17C; (B) structures of oxazole and benzoxazole targets; (C) structures of imidazole and benzimidazole targets. Note: a, N₂CH₂CO₂Et，Rh₂(OAc)₄，CH₂Cl₂；b，LiAlH(OtBu)₃；c，C₆H₅CO₂H, DIAD; d, NaOH, aqueous CH₃OH; e, EDC, HOBt, aminocoumarin.

FIG. 34 shows the quadrant modified by diol-17C synthesis.

FIG. 35 shows a synthetic route to diol-17C. Note: a, C₆H₅CO₂H，DEAD；b，NaBH₄；c，NaH，BrCH₂CH₂Br; d, hydrochloric acid 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de]Anthracene-10-one, Et₃N；e，NaOH，MeOH。

FIG. 36 shows a synthetic route to diol-17C analogs, modified in quadrant 2. Note: a, N₂CH₂CO₂Et，Rh₂(OAc)₄；b，LiAlH(OtBu)₃；c，C₆H₅CO₂H, DIAD; d, NaOH, aqueous MeOH; e, various standard synthetic reactions.

Figure 37 shows an analog of diol-17C, where the amine substituent in the coumarin is modified with substituent X. Note: diol-17C-2, X ═ NH₂(ii) a diol-17C-3, X ═ N (CH)₃)₂(ii) a diol-17C-4, X ═ -NHC (═ O) OC₂H₅(ii) a diol-17C-5, X ═ OCH₃(ii) a diol-17C-6, X ═ -NHCH₂CH₂N(CH₃)₂(ii) a diol-17C-7, X ═ N (CH)₂CH₂)₂NCH₃。

Detailed Description

While the subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process steps may be performed without departing from the scope of the disclosure.

Ranges of values are disclosed herein. The range defines a lower limit and an upper limit. Unless otherwise specified, a range includes all values up to the size of the minimum value (lower limit value or upper limit value) and ranges between values of the range.

As used herein, unless otherwise specified, the term "group" refers to a chemical entity that is monovalent (i.e., has one end that can be covalently bonded to other chemical species, such as methyl or phenyl), divalent, or multivalent (i.e., has two or more ends that can be covalently bonded to other chemical species, such as methylene or phenylene). The term "group" also includes groups (e.g., monovalent groups and multivalent groups, such as divalent groups, trivalent groups, and the like).

As used herein, unless otherwise specified, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-and isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like. The alkyl group may be C₁To C₁₂Alkyl, including all integers and ranges therebetween (e.g. C)₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁Or C₁₂). An aryl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups (alkoxide groups), carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

As used herein, unless otherwise specified, the term "heteroalkyl" refers to a branched or unbranched saturated or unsaturated hydrocarbon group having at least one heteroatom. Examples of suitable heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur and phosphorus, and halogens. Heteroalkyl groups may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

As used herein, unless otherwise specified, the term "aryl" refers to C₅To C₁₂Aromatic or partially aromatic carbocyclic groups, including all integer carbon numbers and carbon number ranges therebetween (e.g., C)₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁Or C₁₂). Aryl groups may also be referred to as aromatic groups. The aryl groups may include polyaryl groups, such as fused rings or biaryl groups. An aryl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, substituents such as halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof. Examples of aryl groups include, but are not limited to, phenyl, biaryl (e.g., biphenyl, etc.), and fused ring groups (e.g., naphthyl, etc.).

As used herein, unless otherwise specified, the term "carbocycle" or "heterocycle" refers to a carbocycle-containing or carbon-containing ring in which one or more carbon atoms are each replaced by a heteroatom. These groups may be non-aromatic or aromatic. The carbocyclic or heterocyclic groups may be saturated or unsaturated, and may have one or more substituents (e.g., hydroxy, alkoxy, thioalkoxy, halogen, etc.), and combinations thereof. Other examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

As used herein, unless otherwise specified, the term "heterocyclic group" refers to a C that includes one or more heteroatoms (e.g., N, O, S) as part of a ring structure₃-C₂₀Cyclic group including all integer carbon numbers and carbon number ranges therebetween (C)₃、C₄、C⁵、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉Or C₂₀). Heterocyclic groups may be substituted or unsubstituted and/or have additional unsaturation. Examples of substituents include, but are not limited toIn the case of halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof. The heterocyclic groups may be fused to carbocyclic groups or to each other. Non-limiting examples of heterocyclic groups include furyl, oxazolyl, isothiazolyl, thiazolyl, tetrahydropyranyl, piperazinyl, dioxanyl, pyrrolidinyl, tetrahydrothienyl, tetrahydrofuranyl, quinuclidinyl, azaadamantyl, decahydroquinolinyl, and the like.

As used herein, unless otherwise specified, the term "heteroaryl" refers to a monovalent monocyclic or polycyclic aryl of 5 to 18 ring atoms, containing one or more ring heteroatoms selected from N, O or S, with the remaining ring atoms being C, including all integer numbers of ring atoms and ranges therebetween (e.g., 5,6,7,8, 9, 10,11,12, 13, 14,15,16,17, or 18). Heteroaryl as defined herein also refers to a polycyclic (e.g., bicyclic) heteroaromatic group wherein the heteroatom is selected from N, O or S. The aromatic groups are optionally independently substituted with one or more substituents described herein. The substituents themselves may be optionally substituted. Examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof. Examples of heteroaryl groups include, but are not limited to, benzothienyl, furyl, thienyl, pyrrolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolinyl, benzopyran, isothiazolyl, thiazolyl, thiadiazolyl, thieno [3,2-b ] groups]Thiophene, triazolyl, triazinyl, imidazo [1,2-b ]]Pyrazolyl, furo [2,3-c ] s]Pyridyl, imidazo [1,2-a ]]Pyridyl, indazolyl, pyrrolo [2,3-c ]]Pyridyl, pyrrolo [3,2-c]Pyridyl, pyrazolo [3,4-c]Pyridyl, benzimidazolyl, thieno [3,2-c ]]Pyridyl, thieno [2,3-c ]]Pyridyl, thieno [2,3-b ]]Pyridyl, benzothiazolyl, indolyl, indolinyl, indolonyl, dihydrobenzothienyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxelohexyl, quinolinyl, isoquinolinyl, 1, 6-naphthyridinyl, benzo [ de ] de]Isoquinolinyl, pyrido [4,3-b ]][1,6]Naphthyridinyl, thieno [2,3-b ]]Pyrazinyl, quinazolinyl, tetrazolo [1,5-a ]]Pyridyl, [1,2,4 ] or a salt thereof]Triazolo [4,3-a]Pyridyl, isoindolyl, pyrrolo [2,3-b ]]Pyridyl, pyrrolo [3,4-b]Pyridyl, pyrrolo [3,2-b]Pyridyl, imidazo [5,4-b ]]Pyridyl, pyrrolo [1,2-a ]]Pyrimidinyl, tetrahydropyrrolo [1,2-a ] s]Pyrimidinyl, 3, 4-dihydro-2H-1 Lambda²-pyrrolo [2,1-b]Pyrimidine, dibenzo [ b, d ]]Thiophene, pyridine-2-ones, furo [3,2-c ]]Pyridyl, furo [2,3-c ]]Pyridyl, 1H-pyrido [3,4-b ]][1,4]Thiazinyl, benzoxazolyl, benzisoxazolyl, furo [2,3-b]Pyridyl, benzothienyl, 1, 5-naphthyridinyl, furo [3,2-b ] and their use as medicaments]Pyridine, [1,2,4 ]]Triazolo [1,5-a]Pyridyl, benzo [ [1,2,3 ]]Triazolyl, imidazo [1,2-a ]]Pyrimidinyl, [1,2,4 ] or their salts]Triazolo [4,3-b]Pyridazinyl, benzo [ c)][1,2,5]Thiadiazolyl, benzo [ c ]][1,2,5]Oxadiazole, 1, 3-dihydro-2H-benzo [ d]Imidazol-2-one, 3, 4-dihydro-2H-pyrazolo [1,5-b][1,2]Oxazinyl, 4,5,6, 7-tetrahydropyrazolo [1,5-a]Pyridyl, thiazolo [5,4-d ]]Thiazolyl, imidazo [2,1-b ]][1,3,4]Thiadiazolyl, thieno [2,3-b ]]Pyrrolyl, 3H-indolyl, and derivatives thereof. Further, when two fused rings are included, heteroaryl groups as defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring.

In one aspect, the present disclosure provides functionalized androstane and dihydrotestosterone compounds. The compounds can be functionalized (e.g., modified) with a coumarin ring group or a coumarin isostere group.

In various examples, the compounds of the present disclosure include 5 α -androstane-3 α,17 β -diol (diol), 5 α -androstane-3 α -ol-17-one, 5 α -androstane-3, 17-dione (5 α -dione), and 5 α -Dihydrotestosterone (DHT), which may be modified with various saturated or unsaturated heterocyclic or carbocyclic rings. In various examples, the compounds can be modified (e.g., functionalized) with coumarin-containing groups or coumarin isostere groups at the C-3 or C-17 positions in these steroids.

The compounds of the present disclosure may have the following structure:

Or a carbonyl group.

Z may be a variety of terminal groups. In various examples, Z is a carbocyclic or heterocyclic group, which may be saturated or unsaturated, and may have one or more substituents (e.g., hydroxy, alkoxy, thioalkoxy, halogen, and the like, and combinations thereof). Non-limiting examples of Z groups include:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the like,

wherein X' is independently selected at each occurrence from hydrogen, alkyl groups, cycloalkyl groups, alkoxy groups, halogens, and combinations thereof. The Z group may have various substituents. Non-limiting examples of substituents include halogens (e.g., -F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl, alkoxide, or phenoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

Various photoactive groups can be used. Photoactive group (R)⁵) Non-limiting examples of (a) include:

and the like.

Androgens having a substituted coumarin attached to C-3 or C-17 are of the general formula. The compound may have the following structure:

wherein R is¹And R²Each at each occurrence is selected from hydrogen and alkyl groups (e.g., methyl groups, ethyl groups, n-propyl and isopropyl groups, etc.); x and Y are each hydroxy or hydrogen, or X and Y comprise a carbonyl group; and Z' is a substituted coumarin ring or a coumarin-isostere. In various examples, R¹And R²Each at each occurrence is selected from hydrogen and alkyl groups (e.g., methyl groups, ethyl groups, n-propyl and isopropyl groups, etc.);x and Y are spiro-fused, substituted coumarin rings or coumarin isosteres; and Z' is hydrogen.

The compound may have the following structure:

wherein R is¹Each at each occurrence is selected from hydrogen and alkyl groups (e.g., methyl groups, ethyl groups, n-propyl and isopropyl groups, etc.); x and Y are each hydroxy or hydrogen; and Z "is an O- (carboxymethyl) oxime attached to a substituted coumarin or coumarin-isostere. In various examples, R¹Each at each occurrence is selected from hydrogen and alkyl groups (e.g., methyl groups, ethyl groups, n-propyl and isopropyl groups, etc.); x and Y are spiro-fused, substituted coumarin rings or coumarin isosteres; and Z "is oxygen.

Representative examples of compounds (I) having a substituted coumarin attached at the C-17 position. Non-limiting examples of compounds of the present disclosure include those of: the group X in compound (I) is a hydroxyl group; y is hydrogen and Z 'is a substituted coumarin wherein the substituent connects the C-17 β oxygen of the steroid to the linker L between the C-4' position of the coumarin ring or coumarin isostere. Examples of such compounds include, but are not limited to, compounds (Ia), (Ib), and (Ic):

wherein X' is independently selected at each occurrence from hydrogen, alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, and the like), cycloalkyl groups, alkoxy groups (e.g., -O (CH)₂)_nCH₃Wherein n is 0,1 or 2), and/or halogen (i.e., -F, -Cl, -Br, and-I) and L is a linker (e.g., a linker group). Examples of linkers include, but are not limited to, the following structures, wherein n and m are independently 1,2,3, 4,5, or 6:

non-limiting examples of compounds of the present disclosure include those of: the groups X and Y in compound (I) are carbonyl groups and Z 'is a substituted coumarin wherein the substituent connects the C-17 beta oxygen of the steroid to the linker L between the C-4' position of the coumarin ring or coumarin isostere. Examples of compounds include, but are not limited to, compounds (Id), (Ie), and (If):

representative examples of compounds (II) having a substituted coumarin attached at the C-17 position. Non-limiting examples of compounds of the present disclosure include those of: the groups X and Y of compound (II) are, respectively, a hydroxyl group and hydrogen, or a carbonyl group or an oxime. Examples of groups include, but are not limited to, compounds (IIa) and (IIb):

in general, representative examples of compounds (I) having a substituted coumarin attached at the C-3 position. Non-limiting examples of compounds of the present disclosure include those of: the groups X and Y of compound (I) are spiro-fused, substituted coumarin ring groups or coumarin isostere groups; and Z' is hydrogen. Examples include, but are not limited to, compounds (Ig), (Ih), and (Ii):

non-limiting examples of compounds of the present disclosure include those of: compound (II) R¹Each occurrence of the group is selected from hydrogen and alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, etc.); the groups X and Y are spiro-fused, substituted coumarin rings or coumarin isosteres; and Z "is oxygen. Examples include, but are not limited to, compounds (Ij), (Ik), and (Il):

in various examples, the compound of the present disclosure is PROTAC. ProTAC may have the following structure: A-L-B, wherein A is E3ligase recruiter (E3 ligand reciever), B is a ligand group for a target protein, and L is a linker covalently linking the E3ligase recruiter and the ligand group.

In one example, the E3ligase recruiter is capable of recruiting proteins, e.g., VHL, CRBN, RNF114, MDM2, DCAF15, DCAF16, SCF, etc., that are capable of ubiquitinating a target protein, such as AR. Non-limiting examples of E3ligase recruitment groups (E3ligase receiver groups) include:

(for example,

(e.g. in

)、

(e.g. in

)、

(e.g. in

)、

(e.g. in

) Or a ligase recruitment group that binds to cerebellum, von hippel-lindau (VHL), or mouse double minute 2(MDM 2).

The compounds of the present disclosure (e.g., PROTAC compounds) can include various linkers (e.g., linking groups). The linker connects E3 to the enzyme recruit and the ligand group of the target protein by a covalent bond (e.g., two or more covalent bonds). Various linkers are known in the art. Non-limiting examples of linkers include: -NH (CH)₂)_nNH-，-C(＝O)(CH₂)_nNH-，-C(＝O)(CH₂)_nC(＝O)-，-NH(CH₂CH₂O)nCH₂CH₂NH-，-C(＝O)(CH₂CH₂O)nCH₂CH₂NH-，-C(＝O)(CH₂CH₂O)nCH₂CH₂C (═ O) -, where n is 2-4 (e.g., 2,3, or 4).

Non-limiting examples of target proteins include protein-associated cancers (e.g., leukemia, lung cancer (e.g., non-small cell lung cancer), skin cancer, upper digestive tract pre-cancerous lesions, prostate cancer, brain cancer, breast cancer, solid tumors, and the like), infectious diseases, inflammatory diseases, immune disorders, sleep disorders, neurodegenerative diseases, and the like, and combinations thereof.

The compounds of the present disclosure are useful for molecular imaging. Compounds suitable for molecular imaging may comprise R as described herein⁵The group may alternatively comprise a spiro-coumarin ring group or both. Non-limiting examples of compounds suitable for molecular imaging (e.g., imaging proliferating cells and/or malignant cells) include:

etc., or combinations thereof.

Specific examples of compounds of the present disclosure include, but are not limited to:

the synthesis of the compounds of the present disclosure may be from commercially available materials. For example, the present disclosure provides methods for preparing compounds (I) and (II) from commercially available materials, wherein a coumarin moiety is attached to the steroid at the C-3 or C-17 position. Non-limiting examples of methods are provided herein. The synthetic routes for these compounds are shown in figures 2,3 and 4. Compounds were prepared by the methods of the present disclosure and characterized by the following structures and purities. In a Varian instrument (¹H. 400 or 500 MHz;¹³C. 100Mz) were determined. High resolution electrospray ionization (ESI) mass spectra were recorded on a Q active mass spectrometer (Thermo Fisher Scientific), Vorteham, Mass.). The resolution was set at 100,000 (at 400 m/z). The sample was introduced by direct injection using a syringe pump at a flow rate of 3 μ L/min. Use of¹³C NMR and precision mass spectrometry determined the purity of the compound. Unless otherwise indicated, compounds are prepared inChromatographic analysis was performed on layer Merck silica gel F254.

The composition may comprise further components. For example, the composition comprises a buffered solution suitable for administration to a subject (e.g., a mammal, such as a human or non-human). An individual may be an object. The buffered solution may be a pharmaceutically acceptable carrier.

The compositions may comprise one or more standard pharmaceutically acceptable carriers. Non-limiting examples of compositions include solutions, suspensions, emulsions, solid injectable compositions dissolved or suspended in a solvent prior to use, and the like. Injections may be prepared by dissolving, suspending or emulsifying one or more active ingredients in a diluent. Non-limiting examples of diluents include distilled water for injection, physiological saline, vegetable oil, alcohol, and the like, and combinations thereof. In addition, the injection may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives and the like. Injections may be sterilized in the final formulation step or prepared by aseptic procedures. The compositions may also be formulated as sterile solid preparations, for example by freeze-drying, and may be sterilized immediately before use or may be dissolved in sterile water for injection or other sterile diluents for use. Non-limiting examples of pharmaceutically acceptable carriers can be found in: ramiden "pharmaceutical Science and Practice" (Remington: The Science and Practice of Pharmacy) (2005), 21 st edition, Philadelphia, PA): WW publishing company (Lippincott Williams & Wilkins).

Non-limiting examples of cancer include leukemia, lung cancer (e.g., non-small cell lung cancer), skin cancer, upper digestive tract precancerous lesions, prostate cancer, brain cancer, breast cancer, solid tumors, and the like, and combinations thereof.

Non-limiting examples of other diseases include infectious diseases, inflammatory diseases, immune disorders, sleep disorders, neurodegenerative diseases, and the like, and combinations thereof.

The methods of the present disclosure can be used to treat various types of cancer, such as hormonal cancer. Non-limiting examples of hormonal cancers include breast, ovarian, uterine or endometrial cancer, prostate cancer, and the like.

The methods of the present disclosure are useful for treating CaP (e.g., androgen-stimulated CaP and CRPC) by inhibiting late oxidoreductase in androgen biosynthesis and/or impairing AR transactivation or AR dimerization. In treating CRPC, the methods described herein can be used to overcome AR-V7-mediated resistance. The methods of the present disclosure may inhibit growth of CaP cells better than methods that include treatment with enzalutamide alone.

The compounds of the present disclosure are useful in methods of treating diseases associated with malignant cells (e.g., CaP) and/or proliferating cells (e.g., benign prostatic hyperplasia or hypertrophy (BPH)). The compounds inhibit the conversion of terminal substrates to DHT or impair AR transactivation or AR dimerization. These compounds may be used in conjunction with or in place of current CaP therapies, such as bicalutamide, enzalutamide or abiraterone.

In various examples, any known methods and routes are used, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intracranial injection. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous administration. Topical and/or transdermal administration is also included.

The methods can be performed in a subject in need of treatment who has been diagnosed with, or is suspected of having, CaP (e.g., a male subject (e.g., an individual) having CaP). The methods may also be performed in subjects who have relapsed or are at high risk of relapse after receiving CaP therapy. An object may be referred to as an individual.

A method of treating a disease (e.g., a cancer, e.g., a hormonal cancer, e.g., a prostate cancer, e.g., a castration-resistant prostate cancer that expresses AR-V7) comprises administering to a subject in need of treatment (e.g., a person in need of treatment) a therapeutic amount of a compound or composition of the present disclosure (e.g., an amount of the compound or composition sufficient to treat the subject), wherein the disease of the subject is treated.

In various examples, the compounds of the present disclosure are used to inhibit the growth of a cell (e.g., a malignant cell, e.g., a cancer cell, e.g., a hormonal cancer cell, e.g., a prostate cancer cell, e.g., a castration-resistant prostate cancer cell expressing AR-V7 or a proliferative cell, e.g., a benign prostate hyperplasia or mast (BPH) cell)). For example, the growth of a cancer cell (e.g., a prostate cancer cell, e.g., a castration-resistant prostate cancer cell that expresses AR-V7) is inhibited by contacting the cancer cell with a compound in an amount (e.g., 1nM to 1mM) and for a time sufficient to cause degradation of an Androgen Receptor (AR) in the cancer cell, wherein degradation of the AR results in inhibition of the growth of the cancer cell. Inhibition of growth may be better than inhibition of cell growth caused by other compounds/treatments known in the art (e.g., treatment with enzalutamide). Inhibition of cell growth refers to any reduction in cell growth/proliferation (e.g., growth/proliferation of cancer cells).

Methods of inhibiting cell growth comprise contacting a cell with a compound of the disclosure or a composition comprising a compound of the disclosure.

The present disclosure provides methods of inducing selective degradation of a target protein. The method of inducing selective degradation of a target protein comprises: i) contacting a cell (e.g., a cell of a subject in need of treatment) with a compound and/or composition of the disclosure, wherein the compound binds to E3ligase and the target protein in the cell.

The subject in need of treatment or the individual in need of treatment may be a human or non-human mammal. Non-limiting examples of non-human mammals include cows, pigs, mice, rats, rabbits, cats, dogs or other agricultural animals, pets, service animals, and the like. In various examples, the subject or individual is a male or has a male reproductive organ. In various examples, the subject or individual has a prostate.

The methods of contacting and/or administering of the present disclosure may be performed in combination with one or more additional agents. The additional agent may be selected from the group consisting of an anti-androgen agent, a 5 alpha-reductase agent, an androgen metabolism inhibitor agent, and combinations thereof. Non-limiting examples of antiandrogens include flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, dilutamide, and the like, and combinations thereof. Non-limiting examples of 5 α -reductase drugs include finasteride, dutasteride, and the like, and combinations thereof. Non-limiting examples of androgen metabolizing drugs include abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and the like, and combinations thereof.

The compounds of the present disclosure are useful as tools for screening small molecule inhibitors of oxidoreductase or small molecule antiandrogens that bind to AR.

In one example, the compounds and compositions are suitable for use in methods involving fluorescence microscopy. Methods involving fluorescence microscopy may be combined with other techniques, such as flow cytometry. Fluorescence microscopy techniques are known in the art. For example, such techniques can be used to monitor cellular activity (e.g., androgen metabolism).

Methods comprising molecular tracking (e.g., intracellular tracking) in a cell (e.g., prostate cell) comprise contacting the cell (e.g., prostate cell) with a compound of the present disclosure and imaging the cell (e.g., prostate cell) at regular intervals (e.g., intervals known in the art) (e.g., imaging using fluorescence microscopy) to monitor activity. In one example, the activity is androgen metabolism.

In one aspect, the present disclosure provides a kit or kit. In various examples, a kit or kit comprises a pharmaceutical formulation comprising any one or any combination of the compounds of the present disclosure. In one example, the present disclosure includes a closed or sealed package containing a pharmaceutical formulation. In various examples, the package includes one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for selling, distributing, or using the pharmaceutical compounds and compositions containing them. The printed material may include printed information. The printed information may be provided on a label, on a paper insert or printed on the packaging material. The printed information may include information identifying the compound in the package, the amount and type of other active and/or inactive ingredients in the composition, and instructions for administering the compound and/or composition. The instructions may include information such as the number of doses taken over a given period of time, and/or information directed to a pharmacist and/or other healthcare provider such as a doctor or patient. The printed material may include indications of the pharmaceutical composition and/or any other pharmaceutical agent provided therein for treating a subject having CaP and/or other disease and/or any condition associated with cancer and/or other disease. In various examples, the kit or kit includes a label describing the kit or kit contents and providing indications and/or instructions regarding the use of the kit or kit contents to treat a subject having any cancer and/or other disease.

In various examples, a kit or kit comprises materials useful for administration to a subject in need of CaP treatment. For example, the kit or kit may contain one or more therapeutic agents, which may be in lyophilized form, optionally a reconstitution medium and instructions for administration. The kit or kit may comprise a single dose or multiple doses.

The steps of the methods described in the various embodiments and examples disclosed herein are sufficient to practice the methods of the present disclosure. The methods described in this embodiment are combinations of steps of the disclosed methods. In another embodiment, the method consists of these steps.

The following statements provide examples of the compounds of the present disclosure, methods of using the compounds of the present disclosure, and uses of the compounds of the present disclosure.

Content 1. compounds having the following structure:

wherein R is¹Is hydrogen or an alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, etc.); r³Selected from the group consisting of carbonyl,

Wherein X is hydroxyl and Y is hydrogen or an alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, etc.) or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or a coumarin isostere group, and L is³Is optional and is a linking group; r⁴Selected from the group consisting of carbonyl,

Wherein L is¹Is a linking group, L₂Is optional and is a linking group, Z is a terminal group comprising a substituted or unsubstituted carbocyclic group or a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted coumarin isostere group, and R is²Is an alkyl group or hydrogen; and R is⁵Is a photoactive group, provided that R³Is a carbonyl group or

Or a carbonyl group.

The compound of claim 1, wherein L¹And/or L²And/or L³Independently selected from:

wherein n is 1,2,3, 4,5 or 6 and m is 1,2,3, 4,5 or 6.

The compound of claim 1 or claim 2, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

wherein X' is independently selected at each occurrence from hydrogen, alkyl groups, cycloalkyl groups, alkoxy groups, halogens, and combinations thereof.

A compound according to any preceding claim, wherein the compound has the structure:

wherein n is 1,2 or 3.

The compound of any of the preceding claims, wherein the compound has the structure:

the compound of claim 1, wherein the compound has the structure:

wherein R is independently selected from L¹-A，R⁵，-OH，-NH₂，-CO₂Et，-CN，-CHO，-SO₃H, and-CO₂H, or three adjacent R groups form a fused ring system (e.g.,

) And n is 1,2,3, or 4, and L¹Is a linking group and A is an E3ligase recruitment group wherein only one R is L¹-A。

The compound of

claim

1 or 6, wherein a is formed from:

a compound as described in context 6, wherein a is formed from a ligase recruitment group that is capable of binding to cerebellum, von hippel-lindau (VHL) or mouse double minute 2(MDM 2).

The compound of any of

clauses

1 or 6, wherein the compound has the structure:

the compound of

claim

1 or 6, wherein the compound has the structure:

the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

The compound of claim 1 or claim 11, wherein the compound has the structure:

wherein n is 1,2 or 3.

The compound of claim 1 or contents 11-12, wherein the compound has the structure:

the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

The compound of claim 1 or claim 14, wherein the compound has the structure:

wherein n is 1,2 or 3.

The compound of claim 1 or 14-15, wherein the compound has the structure:

the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

wherein X' is independently selected at each occurrence from hydrogen, alkyl groups, cycloalkyl groups, alkoxy groups, and/or halogens.

The compound of claim 1 or claim 17, wherein the compound has the structure:

wherein n is 1,2 or 3.

The compound of claim 1 or 17-18, wherein the compound has the structure:

the compound of claim 1, wherein the compound has the structure:

the compound of claim 1 or claim 20, wherein the compound has the structure:

a composition comprising a compound as described in any of the preceding and a pharmaceutically acceptable carrier.

Content 23. the composition of content 22, further comprising one or more additional drugs.

The composition of claim 22, wherein the one or more additional agents are selected from the group consisting of an antiandrogen agent, a 5 α -reductase agent, an androgen metabolism inhibitor agent, and combinations thereof.

Content 25. the composition of any of contents 22-24, wherein the antiandrogen drug is selected from the group consisting of flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, dilutamide, and combinations thereof and/or the 5 α -reductase drug is selected from the group consisting of finasteride, dutasteride, and combinations thereof and/or the androgen metabolism drug is selected from the group consisting of abiraterone acetate, abiraterone acetate particles, ketoconazole, and combinations thereof.

The composition of any of claims 22-25, wherein the compound has the structure:

or a combination thereof.

A method of attenuating Androgen Receptor (AR) transactivation and/or androgen receptor dimerization, comprising: contacting a cell with a compound according to any of contents 1-21 or a composition according to any of contents 22-26 in an amount and for a time sufficient to impair androgen receptor transactivation and/or androgen receptor dimerization.

Content 28. the method of content 27, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, diluxamine, finasteride, dutasteride, or a combination thereof.

Content 29. the method of content 27 or content 28, wherein the compound has the structure:

or a combination thereof.

A method of inhibiting cell growth comprising contacting a cell with a compound as described in any of contents 1-21 or a composition as described in any of contents 22-26 in an amount and for a time sufficient to inhibit cell growth.

Content 31. the method of content 30, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, dilutamide, finasteride, dutasteride, abiraterone acetate particles, ketoconazole acetate, or a combination thereof.

Content 32. the method of content 30 or content 31, wherein the compound has the structure:

or a combination thereof.

Content 33. the method of content 31 or content 32, wherein the cell growth inhibited is malignant cell growth and/or hyperplastic cell growth.

The method of any of claims 31-33, wherein the cell growth (e.g., malignant cell growth and/or hyperplastic cell growth) is a cancer cell growth.

The method of any of claims 31-34, wherein the cell growth (e.g., malignant cell growth and/or hyperplastic cell growth, which can be cancer cell growth) is hormonal cancer cell growth.

The method of claim 35, wherein the hormonal cancer is selected from the group consisting of breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof.

The method of any of clauses 31-35, wherein the cell growth (e.g., malignant cell growth and/or hyperplastic cell growth, which can be cancer cell growth, which can be hormonal cancer cell growth) is prostate cancer cell growth.

The method of any of clauses 31-35 or clause 37, wherein the cell growth (e.g., malignant cell growth and/or proliferative cell growth, which may be cancer cell growth, which may be hormonal cancer cell growth, which may be prostate cancer cell growth) is castration-resistant prostate cancer cells.

The method of any of contents 31-35 or contents 37-38, wherein the cells (e.g., malignant cell growth and/or proliferative cell growth, which may be cancer cell growth, which may be hormonal cancer cell growth, which may be prostate cancer cell growth, which may be castration resistant prostate cancer cells) express AR-V7.

A method of treating a proliferative cell growth disease and/or a malignant cell growth disease in an individual comprising administering to the individual a compound according to any of claims 1 to 21 or a composition according to any of claims 22 to 26, wherein the malignant cell growth disease and/or the proliferative cell growth disease in the individual is treated.

The method of content 40, wherein the malignant cell growth disorder is cancer.

The method of content 40 or 41, wherein the malignant cell growth disorder (e.g., cancer) is a hormonal cancer.

The method of claim 42, wherein the hormonal cancer is selected from the group consisting of breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof.

The method of any of contents 40-43, wherein the malignant cell growth disorder (e.g., cancer, which may be a hormonal cancer) is prostate cancer.

The method of any of contents 40-44, wherein the malignant cell growth disorder (e.g., cancer, which may be a hormonal cancer, which may be prostate cancer) is castration-resistant prostate cancer.

A method as described in any of contents 40-45, wherein cells of the malignant cell growth disease (e.g., a carcinoma, which may be a hormonal carcinoma, which may be a prostate carcinoma, which may be a castration-resistant prostate carcinoma) express AR-V7.

The method of any of aspects 40-46, wherein the compound has the structure:

or a combination thereof.

Content 48. a method of imaging malignant and/or proliferating cells in an individual, comprising: administering one or more compounds according to any of

claims

1 or 6 or 9-10 or 20-21 or a composition according to any of claims 22-26 to an individual, exposing the individual or a portion thereof to electromagnetic radiation, thereby exciting the one or more compounds, detecting the one or more excited compounds, and imaging the individual or portion thereof, wherein malignant cells and/or hyperproliferative cells are imaged.

The method of claim 48, wherein the malignant cell is a cancer cell.

Content 50 the method of content 48 or 49, wherein the malignant cell (e.g., a cancer cell) is a hormonal cancer cell.

The method of any of clauses 48-50, wherein the malignant cells (e.g., cancer cells, which may be hormonal cancer cells) are selected from breast cancer cells, ovarian cancer cells, uterine or endometrial cancer cells, and prostate cancer cells.

The method of contents 48-51, wherein the malignant cell (e.g., a cancer cell, which can be a hormonal cancer cell) is a prostate cancer cell.

The method of contents 48-52, wherein the malignant cell (e.g., a cancer cell, which can be a hormonal cancer cell, which can be a prostate cancer cell) is a castration resistant prostate cancer cell.

The method of contents 48-53, wherein the malignant cell (e.g., a cancer cell, which may be a hormonal cancer cell, which may be a prostate cancer cell, which may be a castration-resistant prostate cancer cell) expresses AR-V7.

Contents 55. the method of contents 48 to 54, wherein imaging is repeated so that the activity of androgen metabolism can be measured.

The method of contents 48-55, wherein the compound has the structure:

or a combination thereof.

The following examples are provided to further illustrate the present disclosure. They are not intended to be limiting in any way.

Example 1

This example provides a description of the compounds of the present disclosure.

Synthesis of Compound (Ia and 17C) as shown below and in FIG. 2, where R₁And R₂Is hydrogen:

ethyl (3-keto-5 α -androstan-17 β -yl) glycolate. 1g (3.44mmol) of androstan-17 beta-ol-3-one and 95mg (0.22mmol, 0.064 eq.) at 0 ℃ with vigorous stirring) Tetraacetic acid dirhodium in 10mL CH₂Cl₂393mg (3.44mmol, 1 eq) of ethyl diazoacetate are added dropwise to the suspension in (1). After 2h at 25 ℃ a further 393mg (3.44mmol, 1 eq) of ethyl diazoacetate are added dropwise and the mixture is stirred for a further 2h (h). CH for the mixture₂Cl₂Diluting, washing with water and brine, and washing with anhydrous MgSO₄Dried and concentrated. The product was purified by column chromatography using 1:3 EtOAc-hexane to give 687mg (53%) of ethyl (3-keto-5 α -androstan-17 β -yl) glycolate as a semi-solid.¹H NMR(CDCl₃,500MHz):δ4.20(q,2H,J＝7.2Hz),4.09(s,2H),3.40(t,1H,J＝8.5Hz),2.42-2.35(m,1H),2.32-2.24(m,2H),2.11-2.00(m,3H),1.96-1.92(m,1H),1.72-1.68(m,1H),1.58-1.26(m,13H),1.21-1.15(m,1H),1.02(s,3H),1.00-0.83(m,2H),0.82(s,3H),0.75-0.69(m,1H).¹³C NMR(CDCl₃,100MHz):δ212.22,171.03,90.02,67.80,60.94,54.09,51.12,46.91,44.89,43.28,38.77,38.36,37.90,35.93,35.38,31.43,28.99,27.76,23.48,21.27,14.42,11.86,11.68.HRMS(ESI)C₂₃H₃₇O₄[MH+]Calculated value 377.2686, measured value 377.2687.

Ethyl (3 β -hydroxy-5 α -androstan-17 β -yl) glycolate. To a solution of 300mg (0.80mmol) of ethyl (3-keto-5 α -androstan-17 β -yl) glycolate in 4mL of THF at-20 ℃ was added dropwise 2.1mL (1.04mmol, 1.3 equiv) lithium tri-tert-butoxyaluminum hydride (0.5M in bis (2-methoxyethyl) ether). The mixture was stirred for 3h, quenched with water, and quenched with CH₂Cl₂Extracting with anhydrous MgSO₄Dried and concentrated. The product was purified by column chromatography using 1: 2 EtOAc-hexane to give 280mg (93%) of ethyl (3 β -hydroxy-5 α -androstan-17 β -yl) glycolate as a white solid. mp 72-74 ℃.¹H NMR(CDCl₃,500MHz):δ4.20(q,2H,J＝7.0Hz),4.10,4.06(ABq,2H,J_AB＝16.2Hz),3.61-3.55(m,1H),3.39(t,1H,J＝8.2Hz),2.05-1.98(m,1H),1.93-1.90(m,1H),1.81-1.77(m,1H),1.72-1.64(m,3H),1.59-1.53(m,4H),1.43-1.36(m,2H),1.32-1.21(m,8H),1.17-1.07(m,2H),0.99-0.82(m,3H),0.81(s,3H),0.79(s,3H),0.65-0.59(m,1H).¹³C NMR(CDCl₃,100MHz):δ170.89,89.94,71.31,67.60,60.72,54.45,51.12,44.87,43.09,38.18,37.86,37.02,35.55,35.29,31.59,31.50,28.56,27.59,23.27,20.88,14.22,12.34,11.66.HRMS(ESI)C₂₃H₃₉O₄[MH+]Calculated value 379.2843, measured value 379.2843.

Ethyl (3 α -benzoyloxy-5 α -androstan-17 β -yl) glycolate. To a solution of 200mg (0.53mmol) (3 β -hydroxy-5 α -androstan-17 β -yl) ethyl glycolate and 129mg (1.06mmol, 2 equivalents) benzoic acid in 3mL anhydrous THF was added 214mg (1.06mmol, 2 equivalents) diisopropyl azodicarboxylate. The solution was stirred at 25 ℃ for 12h, quenched with water, and quenched with CH₂Cl₂Extracting with 10% K₂CO₃The aqueous solution and water were washed with anhydrous MgSO₄Dried and concentrated. The product was chromatographed using 1: 5 EtOAc-hexane (R)_f0.53) to yield 181mg (71%) of ethyl (3 α -benzoyloxy-5 α -androstan-17 β -yl) glycolate as a white solid.¹H NMR(CDCl₃,400MHz):δ8.07-8.05(m,2H),7.58-7.54(m,1H),7.47-7.44(m,2H),5.28(br s,1H),4.20(q,2H,J＝7.2Hz),4.12,4.07(ABq,2H,J_AB＝16.4Hz),3.40(t,1H,J＝8.4Hz),2.05-1.98(m,1H),1.95-1.85(m,2H),1.80-1.52(m,7H),1.45-1.14(m,12H),1.03-0.87(m,2H),0.85(s,3H),0.81(s,3H),0.79-0.75(m,1H).¹³C NMR(CDCl₃,100MHz):δ170.85,165.87,132.69,131.18,129.53(two C),128.32(two C),89.97,70.68,67.63,60.70,54.46,51.16,43.10,40.47,37.84,35.97,35.26,33.24,33.00,31.46,28.24,27.59,26.30,23.23,20.47,14.23,11.68,11.43.HRMS(ESI)C₃₀H₄₃O₅[MH+]Calculated value 483.3105, measured value 483.3108.

N-2' - (2,3,4, 5-tetrahydro-1H, 4H-10-one-11-oxa-3 a-azabenzo [ de)]Anthracenyl) ethyl (3 α -hydroxy-5 α -androstan-17 β -yl) hydroxyacetamide (Ia is also known as diol-17C). To a solution of 160mg (0.33mmol) (3 α -benzoyloxy-5 α -androstan-17 β -yl) ethyl glycolate in 3mL methanol was added 0.66mL (1.33mmol,4 equivalents) of 2N NaOH in water. The suspension was stirred at 50 ℃ for 12h, diluted with water and acidified to pH 2 with 3N HCl solution. The precipitate was collected and dried in vacuo to yield 103mg (89%) of (3 α -hydroxy-5 α -androstan-17 β -yl) glycolic acid, pure enough to be used in the next reaction without further purification. To a mixture of 80mg (0.23mmol) of this acid and 90mg (0.2 mmol)5mmol,1.1 equiv.) of 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de]Anthracene-10-one hydrochloride in 2mL 1:1 MeOH-CH₂Cl₂To the mixture in (1.5) were successively added 66mg (0.34 mmol) of EDC, 46mg (0.34mmol,1.5 equiv.) of HOBt and 81mg (0.80mmol,3.5 equiv.) of triethylamine. The mixture was stirred at 25 ℃ for 12h, diluted with water and diluted with CH₂Cl₂Extracting with anhydrous MgSO₄Dried and concentrated. The product was chromatographed using 1:10 CH₃OH-CH₂Cl₂(R_f0.53) to yield 76mg (54%) of Ia and also named diol-17C as a yellow foam:¹H NMR(CDCl₃,400MHz):δ7.12(s,1H),6.74(t,1H,J＝5.8Hz),5.90(s,1H),4.04(br s,1H),3.95,3.90(ABq,2H,J_AB＝15.4Hz),3.60(q,2H,J＝7.2Hz),3.28-3.23(m,5H),2.92-2.87(m,4H),2.79(t,2H,J＝6.2Hz),2.00-1.88(m,5H),1.78-1.71(m,1H),1.69-1.16(m,17H),1.08-1.01(m,1H),0.95-0.80(m,2H),0.78(s,3H),0.75-0.70(m,1H),0.68(s,3H).¹³C NMR(CDCl₃,100MHz):δ170.61,162.19,153.65,151.44,145.97,121.67,118.18,107.86,107.71,106.92,90.00,69.21,66.49,54.33,51.06,49.96,49.51,43.00,39.12,38.15,37.77,36.15,35.84,35.21,32.19,31.64,31.49,29.02,28.36,27.79,27.66,23.25,21.54,20.65,20.50,20.36,11.77,11.21.HRMS(ESI)C₃₈H₅₃N₂O₅[MH+]calculated value 617.3949, measured value 617.3940.

Example 2

This example provides a description of the compounds of the present disclosure.

Synthesis of the Compound (Id also known as DHT-17C) shown in FIG. 2, where R₁And R₂Is hydrogen:

n-2' - (2,3,4, 5-tetrahydro-1H, 4H-10-one-11-oxa-3 a-azabenzo [ de)]Anthryl) ethyl (3-ketone-5 alpha-androstane-17 beta-yl) hydroxyacetamide. To a solution of 100mg (0.27mmol) (3-keto-5 α -androstan-17 β -yl) ethyl glycolate in 4mL THF was added 0.28mL (0.54mmol,2 equiv.) of 2N NaOH in water.The mixture was stirred at 25 ℃ for 5h and concentrated. The residue was diluted with water and acidified to pH 2 with 3N HCl solution to yield a white precipitate, which was collected to provide 83mg (90%) (3-keto-5 α -androstan-17 β -yl) glycolic acid, which was used in the next step without further purification. To 37mg (0.11mmol) of this acid and 42mg (0.12mmol,1.1 eq) of 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de ]]Anthracene-10-one hydrochloride in 1mL CH₂Cl₂To the mixture in (1.5) were added successively 30mg (0.16mmol,1.5 equivalents) of EDC, 24mg (0.16mmol,1.5 equivalents) of HOBt and 38mg (0.37mmol,3.5 equivalents) of triethylamine. The mixture was stirred at 25 ℃ for 12h, diluted with water and diluted with CH₂Cl₂Extracting with anhydrous MgSO₄Dried and concentrated. 5: 1 EtOAc-hexane (R after two expansions) was used_f0.53) the product was purified by chromatography to give 33mg (51%) Id (also known as DHT-17C) as a yellow foam.¹H NMR(CDCl₃,400MHz):δ7.10(s,1H),6.72(t,1H,J＝6.0Hz),5.87(s,1H),3.93,3.89(ABq,2H,J_AB＝15.2Hz),3.59(q,2H,J＝6.8Hz),3.27-3.22(m,5H),2.91-2.85(m,4H),2.78(t,2H,J＝6.4Hz),2.42-2.21(m,3H),2.09-1.88(m,7H),1.78-1.74(m,1H),1.70-1.64(m,1H),1.59-1.20(m,10H),1.10-1.04(m,1H),0.99(s,3H),0.95-0.80(m,2H),0.73-0.66(m,4H).¹³C NMR(CDCl₃,100MHz):δ211.90,170.49,162.12,153.61,151.43,145.97,121.65,118.19,107.80,107.72,106.89,89.83,69.19,53.77,50.79,49.95,49.50,46.66,44.66,42.99,38.52,38.13,38.07,37.61,35.70,35.11,31.65,31.17,28.73,27.78,27.65,23.28,21.52,21.01,20.64,20.51,11.76,11.48.HRMS(ESI)C₃₈H₅₁N₂O₅[MH+]Calculated value 615.3792, measured value 615.3793.

Example 3

This example provides a description of the compounds of the present disclosure.

Synthesis of Compound (IIa) wherein R is as shown in FIG. 3₁Is hydrogen:

17- (O-carboxymethyloximino)) -5 α -androstan-3 α -ol. A mixture of 537mg (1.85mmol)5 α -androstan-3 α -ol-17-one and 404mg (3.7mmol) carboxymethoxylamine hemichloride in 20mL pyridine was stirred at 80 ℃ for 15 h. The product was cooled and concentrated. Dilute the product with water and add CH₂Cl₂And (4) extracting. The organic layer was washed with brine, anhydrous MgSO₄And (5) drying. The solvent was evaporated and the residue was recrystallized from acetone to yield 652mg (97%) of a white solid: mp 171-.¹H NMR(CDCl₃,400MHz):δ4.57(s,2H),4.05(br s,1H),2.54-2.48(m,2H),1.94-0.82(m,20H),0.91(s,3H),0.79(s,3H).¹³C NMR(CDCl₃,100MHz):δ174.3,172.9,69.8,66.4,54.3,53.8,44.5,39.0,36.1,35.7,34.8,33.9,32.0,31.4,30.9,28.9,28.2,26.1,23.0,20.2,17.1,11.1.

8-hydroxy-2, 3,6, 7-tetrahydro-1H, 5H-benzo [ ij ]]Quinolizine. A mixture of 30g (0.244mol) of m-anisidine and 91.7mL (0.927mol,3.8eq.) of 1-bromo-3-chloropropane is heated at 95 ℃ for 1-2h, at which time an exothermic reaction takes place; stopping heating; the temperature of the mixture peaked at 140 ℃ and 145 ℃. As the temperature begins to drop, heating resumes. The mixture was heated at 140 ℃ and 145 ℃ for 48h and then at 175 ℃ and 180 ℃ for 21 h. The mixture was cooled, diluted with 370mL of concentrated HCl, and quenched by the slow addition of 120mL of water. Once all the solids dissolved, two phase separation was performed. The organic layer was washed with 100mL 10% HCl and the aqueous solutions were combined. The aqueous solution was extracted with diethyl ether to remove unreacted 1-bromo-3-chloropropane. To this acidic aqueous solution was added 200mL of 25M aqueous NaOH solution. The solution was extracted with toluene until the organic phase was no longer colored. The organic phase was over anhydrous MgSO₄Drying and removal of the solvent under reduced pressure gave 18g (39%) of a white solid: mp 126-.¹H NMR(400MHz,DMSO-d₆):δ8.62(s,1H),6.47(d,1H,J＝8.0Hz),5.99(d,1H,J＝8.0Hz),3.01-2.96(m,4H),2.57-2.48(m,4H),1.85-1.79(m,4H).¹³C NMR(100MHz,DMSO-d₆):δ153.0,143.6,126.1,111.9,107.7,103.0,49.6,49.1,26.7,22.1,21.4,21.0.

5- (benzyloxycarbonylamino) -3-oxopentanoic acid methyl ester. Adding 17.1g (30mmol) KOH in 70mL MeOH dropwise at 5-10 deg.C to a solution of 35mL (30mmol) dimethyl malonate in 40mL MeOH over 1hAnd (3) solution. The mixture was stirred at about 25 ℃ overnight. The precipitated potassium methyl malonate was collected and washed with cold MeOH. 7.42g (78mmol,1.04 eq) of MgCl₂And a suspension of 17.6g (113mmol,1.5 eq) of potassium methyl malonate in 110mL anhydrous THF was stirred at 50 deg.C for 4 h. In a separate flask, to a solution of 16.7g (75mmol,1 eq) of N-benzyloxycarbonyl beta-alanine in 50mL of THF at 5 deg.C was added 14.6g (90mmol,1.2 eq) of 1,1' -carbonyldiimidazole. The imidazolide solution was stirred at 25 ℃ for 1 h. The imidazolide solution was added dropwise to the methylmagnesium malonate suspension at 25 ℃. The mixture was stirred for about 12h, concentrated under reduced pressure, and diluted with ethyl acetate. Sequentially using KHSO in ethyl acetate solution₄Aqueous solution, NaHCO₃Aqueous solution and brine. The organic layer was passed over anhydrous Na₂SO₄Drying and concentration gave 18g (98%) of a yellow oil which was used without further purification.¹H NMR(400MHz,CDCl3):δ7.34(br s,5H),5.24(br s,1H,NH),5.07(s,2H),3.72(s,3H),3.45(s,4H),2.80(t,2H,J＝5.2Hz).

8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de]Anthracene-10-one hydrochloride. To a solution of 8.33g (44mmol) of 8-hydroxy-2, 3,6, 7-tetrahydro-1H, 5H-benzo [ ij ]]To a suspension of quinolizine and 10.8g (44mmol) methyl 5- (benzyloxycarbonylamino) -3-oxopentanoate in 132mL toluene was added 88mL (88mmol, 2 equivalents) of a 1M solution of titanium triisopropoxide in hexane. The mixture was heated to reflux for about 12 h. The mixture was used 500mL of CH2Cl₂Diluted and poured into 500mL of a stirred saturated sodium potassium tartrate solution. CH for aqueous layer₂Cl₂And (4) extracting. With anhydrous Na₂SO₄The combined organic layers were dried. The filtered solution was concentrated and the residue was dissolved in a mixture of EtOH and hexane. The mixture was stored at 5 ℃ for 3 days. The resulting precipitate was collected and recrystallized from EtOH to provide 13g (77%) of benzyl and [2- (10-oxo-2, 3,5, 6-tetrahydro-1H, 4H, 10H-11-oxa-3 a-aza-benzo [ de ] as a yellow solid]Anthracene-8-yl) -ethyl]A 1:9 mixture of isopropyl carbamate. To 5.05g (13.6mmol) of the ester mixture was added 12mL of concentrated HCl. The solution was heated at 95 ℃ for 9h, cooled and concentrated in vacuo. The residue was suspended in a mixture of MeOH-acetone and filtered to give 43g (98%) 7 as yellow hydrochloride salt: mp 238-.¹H NMR(400MHz,CD3OD):δ7.25(s,1H),6.00(s,1H),3.36-3.24(m,6H),3.09(t,2H,J＝7.2Hz),2.86(m,4H),2.01(m,4H).¹³C NMR(100MHz,CD3OD):δ164.2,154.0,152.6,146.7,123.0,122.9,121.5,109.7,109.4,109.1,109.0,51.4,50.9,39.8,30.5,28.6,22.4,21.6,21.5.

N-2' - (2,3,4, 5-tetrahydro-1H, 4H-10-one-11-oxa-3 a-azabenzo [ de)]Anthracenyl) ethyl 17- (O-carbamoyloximino) -5 alpha-androstan-3 alpha-ol (IIa). At 0 ℃ at 122mg (0.38mmol) of 8- (2-aminoethyl) -2,3,4, 5-tetrahydro-1H, 4H-11-oxa-3 a-aza-benzo [ de]Anthracene-10-one hydrochloride (7) in 4mL 1:3 MeOH-CHCl₃To the solution were added, in order, 75L (0.42mmol,1.1 equiv.) of N, N-diisopropylethylamine, 139mg (0.38mmol) (6), 206mg (1.5mmol,4 equiv.) of 1-hydroxybenzotriazole hydrate (HOBt) and 205mg (1.07mmol,2.8 equiv. q.) of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC). EDC was added in two equal portions, spaced apart by 30 minutes. The mixture was stirred at 0 ℃ for 1h, concentrated and taken up with 1:10MeOH-CH₂Cl₂(R_f0.39) to yield 134mg (56%) of yellow foamy IIa.¹H NMR(CDCl₃,400MHz):δ7.13(s,1H),6.6(t,1H,J＝6.4Hz),5.87(s,1H),4.46(s,2H),4.03(br s,1H),3.65-3.59(m,2H),3.28-3.23(m,4H),2.92-2.85(m,4H),2.79(t,2H),2.46-2.33(m,2H),1.99-1.94(m,4H),1.82-0.73(m,20H),0.84(s,3H),0.79(s,3H).¹³C NMR(CDCl₃,100MHz):δ173.3,170.8,162.3,153.9,151.4,146.1,121.8,118.4,107.8,107.4,106.9,72.6,66.3,54.4,53.9,50.0,49.5,44.5,39.1,38.1,36.2,35.9,34.9,34.1,32.1,31.9,31.5,29.0,28.4,27.8,26.1,23.2,21.6,20.7,20.6,20.3,17.3,11.3.HRMS(ESI)C₃₈H₅₂N₃O₅[MH+]Calculated value 630.3901, measured value 630.3899.

Example 4

This example provides a description of the compounds of the present disclosure.

Synthesis of Compound (IIb) wherein R is shown in FIG. 3₁Is hydrogen:

n-2' - (2,3,4, 5-tetrahydro-1H, 4H-10-one-11-oxa-3 a-azabenzo [ de)]Anthracenyl) ethyl 17- (O-carbamoyloximino) -5 α -androstan-3-one (IIb). To 30mg (0.048mmol) of IIa in 1mL 1:1 DMSO: CH₂Cl₂To the solution in (1) were added 24mg (0.24mmol, 5 equivalents) of triethylamine and 23mg (0.14mmol,3 equivalents) of SO in that order₃-pyridine complexes. The mixture was stirred at 25 ℃ for 5h with CH₂Cl₂Diluted, washed with brine and water, over anhydrous MgSO₄Dried and concentrated. Chromatography on silica gel using 1:10 CH₃OH-CH₂Cl₂(R_f0.56) to yield 16mg (54%) of IIb as a yellow foam.¹H NMR(CDCl₃,400MHz):δ7.10(s,1H),6.46(t,1H,J＝5.8Hz),5.87(s,1H),4.46(s,2H),3.69-3.57(m,2H),3.28-3.23(m,4H),2.91-2.86(m,4H),2.79(t,2H,J＝6.2Hz),2.50-2.24(m,5H),2.11-1.94(m,5H),1.87-1.73(m,3H),1.65-1.60(m,1H),1.57-1.26(m,8H),1.18-1.12(m,1H),1.02-0.92(m,4H),0.88(s,3H),0.83-0.72(m,1H).¹³C NMR(CDCl₃,100MHz):δ211.69,172.96,170.60,162.09,153.62,151.42,145.99,121.64,118.25,107.67,107.33,106.82,72.55,53.81,53.61,49.94,49.49,46.58,44.63,44.41,38.46,38.11,37.71,35.76,34.74,33.97,31.81,31.11,28.69,27.78,26.09,23.20,21.51,20.92,20.62,20.51,17.22,11.45.HRMS(ESI)C₃₈H₅₀N₃O₅[MH+]Calculated value 628.3745, measured value 628.3741.

Example 5

This example provides a description of the compounds of the present disclosure.

Synthesis of the Compound shown in FIG. 4 (Ig also known as DHT-3C), wherein R₁And R₂Is hydrogen:

(3R,5S,10S,13S,17S) -17-hydroxy-10, 13-dimethyl-1, 2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16, 17-docosano-7 ' H,11' H-spiro [ cyclopentane [ a ]]Phenanthrene-3, 4' -pyrido [3,2,1-ij]Pyrido [4', 3': 4,5]Pyrano [2,3-f ]]Quinolines]-5'(1' H) -keto hydrochloride (Ig). To a solution of 66mg (0.21mmol, 1 eq) of 9- (2-aminoethyl) -2,3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-f ]]Pyrido [3,2,1-ij]To a suspension of quinolin-11-one hydrochloride in 2mL of absolute ethanol was added 50mg (0.17mmol, 1 eq.) of DHT. To this suspension in a sealed tube was added 0.2mL of concentrated HCl and the mixture was stirred at reflux for 48 h. The suspension became a clear solution within the first hour of heating and precipitation of the desired product then occurred. The reaction was quenched by the addition of about 3mL of water and the precipitate was collected by filtration to provide 73mg (92%) of Ig. Additional purification was achieved by recrystallization from methanol:¹H NMR(400MHz,DMSO-d₆)9.41-9.13(m,2H),7.14(s,1H),4.43(br s,1H),3.45(t,2H),3.29-3.22(m,4H),3.12-3(m,2H),2.82-2.64(m,4H),2.57(t,J＝14.2Hz,1H),1.96-1.8(m,4H),1.8-1.7(m,2H),1.7-1.55(m,4H),1.56-1.41(m,4H),1.42-1.3(m,3H),1.28-1.06(m,5H),1.05-0.77(m,7H),0.65(s,3H).¹³C NMR(101MHz,DMSO-d₆)158.61,149.23,147.42,145.69,121.59,118.47,114.52,106.22,104.7,80.04,58.69,52.91,50.82,49.16,48.65,42.59(two C),36.7,35.26,35.05,34.83,32.41,31.64,31.17,29.84,27.47,27.09,25.4,23.05,22.54,20.88,20.12,19.96,19.69,11.42,11.37.HRMS(ESI)C₃₆H₄₉N₂O₃[MH+]calculated 557.3738. found 557.3744 suspension of hydrochloride salt of desired product in dichloromethane and NaHCO₃Is washed with a saturated aqueous solution. The dichloromethane layer was passed over anhydrous Na₂SO₄Dried, filtered, concentrated and chromatographed on silica gel using 1:10 methanol-dichloromethane (R)_f0.55) to yield the free base form of Ig:¹H NMR(400MHz,DMSO-d₆)7.03(s,1H),4.4(d,J＝4.8Hz,1H),3.48-3.37(m,1H),3.2(q,J＝5.6Hz,4H),2.84(t,J＝5.7Hz,2H),2.7(q,J＝6Hz,4H),2.58(t,J＝5.6Hz,2H),2.52-2.43(m,1H),2.32(t,J＝13Hz,1H),1.95-1.76(m,6H),1.75-1.67(m,1H),1.66-1.54(m,2H),1.54-1.43(m,2H),1.39-1.26(m,4H),1.25-1.03(m,5H),1-0.75(m,7H),0.74-0.64(m,1H),0.62(s,3H).¹³C NMR(101MHz,DMSO-d₆)159.5,149.38,149.08,144.58,122.52,121.11,117.68,108.18,104.87,80.11,53.89,53.82,50.79,49.16,48.67,42.59,39.94,36.76,36.13,35.69,35.28,35.25,33.32,31.48,29.88,28.18,27.84,27.13,26.3,23.11,21.16,20.28,20.22,19.88,11.61,11.39.HRMS(ESI)C₃₆H₄₉N₂O₃[MH+]calculated value of 557.3738, measured value of 557.3738, anal.C₃₆H₄₈N₂O₃Calculated C, 77.66; h, 8.69; n,5.03, measurement C, 77.53; confirmation of C-3R stereochemical assignment in H,8.62, N,4.95. Spirocyclic DHT adduct Ig relies on two-dimensional 1H-13C Heteronuclear Single Quantum Coherence (HSQC), gradient correlation spectroscopy (gCOSY), and two-dimensional rotating frame NOESY (2D ROESY) experiments. The resonance of the protonated amine in the spiro system occurs at 9.24ppm in the 2D ROESY spectrum and is the starting point for stereochemical partition at C-3. Ammonium group (NH) at C-3₂ ⁺) By DMSO-d₆D in (1)₂And determining by an O exchange experiment. The correlation of the ROESY spectra between this ammonium group and the individual C-1 α, C-2 α, C-4 α and C-5 α protons (FIG. 5) confirms the 3 α orientation of the ammonium group in Ig.

Example 6

This example provides a description of the compounds of the present disclosure.

Synthesis of Compound (Ij) wherein R is shown in FIG. 4₁Is hydrogen:

(3R,5S,10S,13S) -10, 13-dimethyl-1, 2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15, 16-docosahydrocarbono-7 'H,11' H-spiro [ cyclopentane [ a ] a]Phenanthrene-3, 4' -pyrido [3,2,1-ij]Pyrido [4', 3': 4,5]Pyrano [2,3-f ]]Quinoline e]-5',17(1' H,2H) -dione. (Ij). The procedure for the preparation of Ig was repeated using 5 α -androstane-3, 17-dione to give Ij (74%), which was purified using hot methanol trituration.¹H NMR(400MHz,DMSO-d₆)δ7.04(s,1H),3.21(q,J＝5.5Hz,4H),2.85(t,J＝5.8Hz,2H),2.71(q,J＝6.1Hz,4H),2.58(t,J＝5.7Hz,2H),2.46-2.30(m,3H),2.08-1.95(m,1H),1.93-1.78(m,5H),1.74(dd,J＝12.7,3.3Hz,1H),1.7-1.59(m,2H),1.59-1.42(m,3H),1.4-1.32(m,2H),1.31-1.18(m,4H),1.18-1.08(m,3H),1.02-0.96(m,1H),0.95(s,3H),0.79(s,3H),0.79-0.7(m,1H).¹³C NMR(101MHz,DMSO-d₆)δ219.89,159.52,149.46,149.08,144.61,122.46,121.13,117.71,108.15,104.87,53.87,53.64,50.83,49.16,48.66,47.14,36.12,35.77,35.32,34.63,33.23,31.45,30.65,28.01,27.8,27.12,26.28,21.38,21.15,20.27,19.86,13.49,11.57.

Example 7

This example provides a description of the biological activity of (((3R,5S,10S,13S,17S) -3-hydroxy-10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) oxy) -N- (2- (11-oxo-2, 3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-9-yl) ethyl) acetamide described in this disclosure as diol-17C (also known as compound Ia).

CaP cells survive and grow in ADT due to intratumoral synthesis of DHT and AR hypersensitivity. Our group and others previously demonstrated that AR is transactivated by picomolar levels of DHT, CRPC produces sufficient DHT for AR transactivation, and CRPC efficiently metabolizes diols to DHT. Sufficient tissue levels of DHT were determined in CRPC for AR transactivation, resulting in the reuse of abiraterone and FDA approval for treatment of advanced CaP. The front and back door approach to DHT has not been valued until recently. Diol is the direct precursor of DHT in the major posterior pathway (fig. 1), and preclinical studies have demonstrated conversion of diol to DHT using castrate-relapsed CWR-R1 human CaP xenografts. Four 3 α -oxidoreductases that metabolize diol to DHT immunostain Tissue Microarrays (TMAs) constructed from androgen-stimulated benign prostate (AS-BP; n-36) and CaP (AS-CaP; n-36) and CRPC (n-36) (fig. 8). CaP cell pellets analyzed using LC-MS/MS showed that 3 α -oxidoreductase expression increased DHT production (FIG. 9).

The 3 α -oxidoreductase inhibits the conversion of diols to DHT: diols were converted to DHT in CRPC xenografts and four 3 α -oxidoreductases were present in clinical specimens, suggesting that diols are likely to be metabolized to DHT in advanced CaP or CRPC patients. In vitro administration resulted in transactivation of AR-luciferase (fig. 10), which provided the opportunity to examine the inhibitor. The constraint-based polyprotein alignment tool (COBALT) showed that the four 3 α -oxidoreductases share the active site, catalytic amino acid residues. LC-MS/MS confirmed that the four 3 α -oxidoreductases converted diols to DHT and androsterone to 5 α -dione. The 3 alpha-oxidoreductase catalytic amino acid residues were mutated using site-directed mutagenesis. LC-MS/MS showed that single, double or complete catalytic deletion of common catalytic amino acids impairs enzyme activity. Furthermore, the impaired catalytic mutant combined with dutasteride reduced DHT levels in CaP cell lines more than dutasteride alone (figure 11). The experiment was repeated using LAPC-4 cells transfected with RDH16 targeting siRNA. The DHT ELISA showed that knock-down of RDH16 expression decreased LAPC-4DHT levels (fig. 12). Growth of VCaP (figure 13) and LAPC-4 cells (data not shown) was assessed after catalyzing impaired RDH16Y176F, K180R mutant and expression treated with dutasteride.

Identification of 3 α -oxidoreductase inhibitors: the antitumor activity of various non-steroidal compounds is tested by screening PC-3CaP cells. Coumarin-labeled androgens were developed to track changes in androgen metabolic pathways in response to castration or androgen metabolic inhibition using in vitro and in vivo CaP cell models. Cell viability and AR-regulated gene transcription were assessed using (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT.) data were collected on

days

0 and 6, data expressed as relative percent increase (mean 6 days-mean 0 days/mean 0 days). CaP cell lines were cultured for 6 days in SFM alone or SFM containing 20nM diol or 20nM diol-17℃ MTT showed that diol-17C treatment impaired CaP cell growth (FIG. 14).

ImageStream(ImageStream^XMKII flow cytometer [ Amnis, Seattle, Washington State)]) Is a hybrid technology, combines the statistical capability of flow cytometry and the imaging capability of a fluorescence microscope, and is suitable for simultaneously researching the positioning and the movement of the diol-17C compartment. ImageStream Data were normalized to single stain controls and analyzed using Image Data Exploration and Analysis Software (IDEAS; Amnis). By pixel-by-pixel comparisonThe similarity score was measured for two images of the same cell. If the similarity score is found>0, then the two objects (RDH16-YFP and diol-17C) are co-located. diol-17C (blue) was co-localized (white dotted signals) with RDH16-YFP (color changed to green in IDEA for comparison) and DRAQ5 in the nucleus of PC-3-RDH16-YFP cells (FIG. 15A.1 single and merged fluorescence images; 15A.2 upper right quadrant highly similar), but non-SFM-treated PC-3RDH16-YFP (FIG. 15B.1 merged image without dotted signals; 15B.2 upper right quadrant highly similar). Competition studies using ImageStream showed that diol and diol-17C compete for RDH16-YFP in PC-3 cells stably expressing RDH16-YFP (FIG. 16).

LC-MS/MS was performed using CaP cells treated with diol-17C to ensure that the cells did not produce free diol and orphan coumarin. No orphan fluorophore was detected. Addition of coumarin did not impair the 3 α -oxidoreductase metabolism of diol-17C to DHT-17C (FIG. 17).

Example 8

This example provides a description of the biological activity of (((5S,10S,13S,17S) -10, 13-dimethyl-3-oxohexadecahydro-1H-cyclopenta [ a ] phenanthren-17-yl) oxy) -N- (2- (11-oxo-2, 3,6, 7-tetrahydro-1H, 5H, 11H-pyrano [2,3-f ] pyrido [3,2,1-ij ] quinolin-9-yl) ethyl) acetamide described in this disclosure as DHT-17C (also known as compound Id).

Androgen-sensitive AR-positive LAPC-4 and VCaP, castration relapse and AR-positive CWR-R1 and CWR-22rv1(22rv1), and AR-negative PC-3 and DU145 cells were transfected with an empty pCMV expression plasmid (control) or pCMV plasmid encoding wild-type AR (GFP; AR-GFP) with a C-terminal green fluorescent protein marker. AR positive CaP cell lines were transfected with AR-GFP to assess whether DHT-17C or DHT-3C co-localized with AR and translocated to the nucleus. The fluorescent coumarin moiety was moved from the C-17 to the C-3 position (DHT-3C, also known as Compound Ig) to test the effect of coumarin on antiandrogenic activity. The data indicate that the location of the coumarin moiety on DHT is critical for distinguishing DHT-like from antiandrogen-like activity.

AR positive Cap cell lines were treated with androgen-free, serum-free complete medium (SFM) alone or SFM alone containing 20nM or 50nM fluorescent androgen or C for 16 hours. Time points were selected to measure differences in the uptake of fluorescent androgen and nuclear or cytosolic localization of fluorescent androgen and/or AR-GFP. ImageStream analysis indicated that DHT-17C and DHT-3C were taken up by VCaP, LAPC-4, CWR-R1, 22Rv1, PC-3 and DU145 cells (data reported for VCaP and PC-3-AR-GFP, which represent all cell lines studied). DHT-17C (blue) or DHT-3C co-localized with AR-GFP (green) and the nuclear marker DRAQ5 (red) in the nuclei of VCaP-AR-GFP and PC-3-AR-GFP cells (white dotted signals) (FIG. 18)

DHT-3C (also known as Compound Ig) is co-localized with AR-GFP and DRAQ5 (FIG. 18). DHT-3C was used as a negative control for DHT-17C (also known as Compound Id) and indicated that DHT-17C is an inhibitor of AR-LBD, while DHT-3C retained DHT activity (as described above).

No nuclear localization of DHT-17C or DHT-3C was observed in AR or AR-GFP negative PC-3 cells, indicating that the nuclear localization of DHT-17C or DHT-3C is AR dependent. ImageStream study was repeated using VCaP and PC-3 cells stably expressing AR-V2 (lacking the N-terminal splice variant) and C-terminal GFP (AR-V2-GFP). ImageStream analysis showed that DHT-17C and DHT-3C co-localized with DRAQ5 and AR-V2-GFP (FIG. 18). The ImageStream data was confirmed by Immunoprecipitation (IP) studies using DHT-targeting antibodies.

ImageStream was used to conduct competition studies between DHT-17C and enzalutamide against AR-GFP or AR-V2 to determine that DHT-17C targets AR-LBD. ImageStream data confirmed that DHT-17C and enzalutamide compete for AR-LBD of wild-type AR and AR-V2 (FIG. 19).

Vcap, CWR-R1 and PC-3 cells were treated with 10. mu.M Complete Medium (CM), Serum Free Medium (SFM), bicalutamide (Bic), 20. mu.M coumarin C, 10nM DHT, 20nM DHT-17C and 20nM DHT-3C for six days (FIG. 20). MTT assay analysis showed that treatment with DHT-3C for 6 days promoted the growth of VCaP, LAPC-4 and LNCaP cells compared to DHT, as expected by DHT (data not shown) and a surrogate for CWR-R1 cell growth (figure 20). In contrast, DHT-17C treatment impaired VCaP and CWR-R1 cell growth and enhanced the effect of bicalutamide (FIG. 20; p value < 0.05). Similar responses were generated using MTT experiments with LAPC-4, LNCaP and 22Rv1 (data not shown; p value < 0.05). DHT-17C did not impair cell growth in AR negative PC-3 (FIG. 20), DU145 and non-CaP cell lines (CV-1 monkey kidney and 293 human kidney cell lines). MTT assays were performed to compare cell growth damage of VCaP cells treated with DHT-17C and enzalutamide. Cells were treated with DHT-17C at 1nM, 12nM, 20nM, 50nM or 100nM concentration or enzalutamide at 1. mu.M, 12. mu.M, 20. mu.M, 50. mu.M or 100. mu.M concentration. DHT-17C at nM concentration impaired VCaP cell growth to a similar extent as enzalutamide at μ M concentration (FIG. 21). MTT using enzalutamide (20uM) or DHT-17C (50nM) treated mutant AR and AR-V7 positive CWR-R1 cells supported VCaP MTT data (FIG. 22).

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 24 hour treatment of untransfected VCaP, LAPC-4, LNCaP, CWR-R1, and 22rv1 cells with DHT or DHT-3C resulted in similar induction of expression of the AR regulatory gene KLK2 (FIG. 23; represented by CWR-R1). In contrast, DHT-17C treatment did not induce KLK2 transcript formation in any of the five AR positive CaP cell lines (FIG. 23; represented by CWR-R1), indicating that the fluorophore placement site on the DHT molecule, rather than the presence of C, is responsible for the lack of induction of KLK 2. The completion of these studies revealed that DHT-17C has anti-androgen like properties, but led to another unexpected finding.

Example 9

DHT-3C is an alternative to DHT and can be used as a vehicle for molecules that disrupt AR transactivation or even degrade AR.

ImageStream was used to evaluate the cellular uptake of DHT-3C (aka Compound Ig) in FIG. 4 by AR-negative PC-3 cells with empty plasmids, PC-3 cells stably expressing AR-GFP (PC-3-AR-GFP), VCaP cells with empty plasmids, VCaP cells stably expressing AR-GFP (VCaP-AR-GFP), or VCaP stably expressing AR170LBD with only splice variant V2(AR-V2-GFP) to confirm the DHT-like activity exhibited by DHT-3C. Serum free complete medium (SFM)27 was used to remove the androgen cells. PC-3 cells containing the empty plasmid were treated with SFM, SFM with C, or SFM with DHT-3C, respectively. The cells showed minimal autofluorescence and did not produce nuclear fluorescence consistent with C or DHT-3C. SFM-treated PC-3AR-GFP cells showed no autofluorescence. SFM-treated PC-3AR-GFP cells with C demonstrated no nuclear co-localization between C and AR-GFP (FIGS. 24A.1 and A.2). DHT-3C treated PC-3-AR-GFP showed nuclear co-localization between DHT-3C and AR-GFP (FIGS. 24B.1 and B.2). C-treated VCaP stably expressing AR-GFP did not produce nuclear co-localization between C and AR-GFP (FIGS. 24C.1 and C.2). DHT-3C treated VCaP stably expressing AR-GFP demonstrated co-localization between DHT-3C and AR-GFP (FIGS. 24D.1 and D.2). DHT-3C treated VCaP stably expressing AR-V2-GFP showed that DHT-3C co-localized with AR LBD (FIGS. 24E.1 and E.2).

ImageStream data showed that DHT-3C was taken up by the CaP cell line and co-localized with AR in the nucleus. MTT assay was used to assess whether treatment with DHT-3C during androgen deprivation promoted growth of CaP cells. AR positive androgen sensitive VCaP, LAPC-4, and LNCaP; AR positive castration-relapsed CWR-R1 and CWR22rv 1; and AR negative PC-3 and DU145 CaP cell lines were treated for six days under five sets of conditions: intact cell Culture Medium (CM) to establish baseline CaP cell growth; SFM mimics androgen deprivation; SFM with only C as negative control to ensure that C alone does not affect CaP cell growth; SFM with DHT as positive control; and SFM with DHT-3C. SFM with DHT-3C stimulated growth in 3 of 5 cell lines (FIG. 25).

Taken together, these data indicate that increased growth of CaP cells is AR dependent and stimulated by DHT or DHT-3C as a replacement for DHT.

Additional data suggest that DHT-3C, like DHT itself, affects AR-regulated gene transcription induction, consistent with the finding that C alone has no effect on AR-regulated transcript induction, and consistent with a computational model in which the coumarin moiety in DHT-3C is predominantly outside the AR-LBD binding pocket. Western blot analysis confirmed that DHT-3C and DHT treatment produced similar AR-regulated protein expression levels (FIG. 26).

In summary, DHT-3C (also known as Compound Ig) in FIG. 4 acts as a true substitute, and its replacement by other ligands (displacement) provides a means to evaluate anti-androgens or to deliver small molecules to disrupt AR transactivation or even degrade AR.

The use of gene "knockdown" techniques to down-regulate AR expression is clinically challenging due to low cellular uptake of oligonucleotides and technical difficulties involved in delivery to the desired target. Furthermore, the recently reported development of SNIPER reagents and hydrophobic labeling methods for AR degradation (HyT) represent an experimental tool for discovery of biology in current performance, rather than a viable therapeutic strategy. People ask that the micromolar curative effect reported by the people is far higher than the current level of the anti-tumor drugs; there are also some problems with undesirable, off-target effects. On the other hand, targeted AR protein degradation induced by proteolytic targeting chimeras (PROTAC) through the ubiquitin-proteasome system (UPS) represents an attractive alternative to the well-designed DHT-3C.

Proteolytic targeting chimera (PROTAC) reagents have three components: a ligand that binds to the target protein, an E3ligase "recruiter" that facilitates ubiquitination of the target protein, and a spacer that links the ligand and the recruiter. Several studies describe the development of PROTAC, which induces degradation of AR proteins by linking various ligands of AR to other ligands that can recruit E3 ubiquitin ligase. For example, one of the studies reported chemical coupling of a selective AR modulator (SARM) with nanomolar affinity for AR to nutlin, a known MDM2E 3ligase ligand. This approach yielded SARM-nutlin PROTAC, which reduced AR levels in HeLa cells in a proteasome-dependent manner, but only at a concentration of 10. mu.M. Two additional studies describe the development of DHT-based PROTAC by linking DHT to ligands for recruitment of SCF β -TRCP and ligands for the canonical-hippel-lindau (VHL) E3 ligase. The PROTAC thus produced shows proteasomal degradation of the AR protein, but only at relatively high concentrations of 10-25 μ M. In addition, the procac based on SCF β -TRCP is not cell permeable and must be microinjected.

In summary, the reported drawbacks of DHT-based PROTACs in terms of potency and cell permeability are the nature of the chemical entities used to target the AR, and thus there is a continuing and unmet need for improved PROTAC therapies. DHT-3C is used as a powerful substitute of DHT, and provides a platform for developing a new PROTAC reagent. For example, modification of the aniline group in compound Ii with a linker and appropriate E3ligase recruits will provide a new and effective family of PROTAC reagents. Modification of the aniline group in compound Ii or C-3O- (carboxymethyl) oxime derivatives of DHT itself with a photoactive group (fig. 29), including diaziridines, benzophenones or perfluorinated aryl azides, can provide a route for the definitive cross-linking of these drugs to the AR or to late enzymes in the pathway leading to DHT and new photodynamic/thermotherapy for late CaP or CRPC patients.

Example 10

The following examples describe the use of compounds Ij and Ig.

Computational models provide information on binding of coumarin-modified androgens to the active site of late enzymes in the biosynthetic pathway that converge on DHT, or binding of coumarin-modified androgens to AR-LBD.

In the former case, computational models of the binding of compound Ij to AKR1C3 (also known as 17 β -hydroxysteroid dehydrogenase-5) show that the compact nature of this fluorescent androgen Ij does not interfere with binding to the active site (fig. 6). The X-ray structure of the ligand binding domain in human AKR1C3(PDB: 1XF0) with a 5 α -diketone (FIG. 6A) was chosen as a template to mimic the binding of compound Ij (also known as 5 α -diketone-F) (FIG. 6B). The original enzyme constructs were downloaded from the RCSB protein database and prepared for docking using the Autodock tool. Compound Ij was docked using Autodock Vina to the position occupied by the 5 α -diketone in AKR1C 3. The binding posture of Ij obtained from Vina highly overlaps with the binding morphology of 5 α -diketones. The binding gesture is further refined by performing a series of energy minimization processes. Briefly, the AMBER14SB force field and the second generation universal AMBER force field (gaff2) were used for protein and ligand, respectively. The partial charge for Ij was generated by the Antechamber9 program in AMBER 18 using the AM1-BCC model. Two blending schemes using 2500-step steepest descent minimizationThe minimization process, followed by conjugate gradient minimization, until a maximum of 2500 iterative steps is reached or the convergence criterion is met (root mean square of energy gradients is less than

). In the first step of minimization, the protein atoms are used

The force constant of (c). The second minimization step involves 1000 steps of steepest descent minimization followed by 1500 steps of conjugate gradient minimization, which is not limiting to either ligand or protein atoms. In summary, compound Ij adopts the same equilibrium as the naturally occurring ligand 5 α -diketone. The BCD ring of compound Ij is inserted into the SP1 binding pocket in the same manner as the 5 α -diketone, and the C-18 and C-19 angular methyl groups of the 5 α -diketone and Ij project into the oxyanion hole of 17 β -HSD5, as defined by Y55, H117 and NADP⁺And (4) surrounding. Hydrogen bonding to S129 further stabilizes the observed binding pattern to Ij. These binding characteristics indicate that compound Ij has a binding mode that matches that of the 5 α -diketone itself. In a similar manner, compound Ij occupies the same binding pocket as the previously described inhibitor 3-carboxamide-1, 3,5- (10) -estratriene-17R-spiro-2- (5, 5-dimethyl-6-oxo) tetrahydropyran (EM1404), 3-carboxamide-1, 3,5- (10) -estratriene-17R-spiro-2- (5, 5-dimethyl-6-oxo) tetrahydropyran binds to AKR1C3(PDB:1ZQ 5).

In the latter case, computational models of binding of compound Ig (also known as DHT-3C) and AR indicate that the BCD ring of compound Ig is inserted into the AR-LBD binding pocket (FIG. 7). The X-ray structure of human AR-LBD complexed with DHT (PDB:2ama) was chosen as a template to mimic AR. The initial structure of AR (694-919) was generated using SWISS-MODEL with the addition of the missing atoms. Compound Ig was added to the site of DHT to create its complex with AR. The composite was carefully refined by performing a series of energy minimization processes and constrained MD simulations. Briefly, the AMBER14SB force field and the second generation universal AMBER force field (gaff2) were used for protein and ligand, respectively. Adding TIP3P water molecule as solvent, using AmberTools18 as soluteAtomic distance from the boundary of the rectangular frame at least

A counter ion, i.e. a chlorine atom, is added to neutralize the net charge of each system. The long-range electrostatic interactions are processed by a Particle Mesh EWald (PME) algorithm, and the non-bonding cutoff for real-space interactions is set to

Two-step minimization was performed using a mixed approach of 8000-step steepest descent minimization and conjugate gradient minimization until a maximum of 2000 iteration steps was reached or a convergence criterion was met (root of energy gradient 291 mean square is less than

In the first minimization procedure, the ligand and protein atoms were applied except for residues 711, 752 and 759-771

The force constant of (c); in the second minimization procedure, the protein framework atoms were applied except for residues 711, 752 and 759-771

The force constant of (c). The system heats linearly from 0 to 303.15K over a 50ps period while constraining all heavy atoms in the NVT ensemble (force constant is

) Then the langevin thermostat was used to balance 325ps in the NPT (P1 atm and T303.15K) set, gradually decreasing the force constant from 10 to

Finally, a 40ns production run was performed in the NPT (P1 atm and T303.15K) pool using Amber12 and at residue 694-759 and 771-919 (force constant of

). The SHAKE algorithm is used to suppress covalent bonds between heavy atoms and hydrogen atoms, with a time step set to 2fs, with snapshots saved every 8 ps. The RMSD converged well in the second half of the simulation, with the last 20ns RMSD fluctuation being less than

The chimera was used to cluster the conformations of the last 20ns trajectory and the top conformation was selected and subjected to the minimization procedure described above (step 2). The final conformation was used for further analysis. Modeling has shown that the hairpin domain (759-771) of the AR is "opened" approximately from its initial position

To accommodate compound Ig. Compound Ig bound to AR-LBD in a similar binding pattern as compound Ig. Compound Ig forms two hydrogen bond interactions with N705 and T877 and produces favorable van der waals interactions with residues L701, F876, M780, L873, L704, M742, W741 and L707 (fig. 7B). In addition to these features, the coumarin moiety of compound Ig also forms hydrophobic interactions with F764, M749, Y763, and R752, and hydrogen-bonding interactions with Q711. These binding characteristics indicate that compound Ig binds AR in a more favorable manner than DHT.

In summary, computational models indicate that successful binding of compounds Ij and Ig will occur to the enzymatic binding pocket of AKR1C3 or AR-LBD, respectively, even though the fluorescent labels attached to the 5 α -diketone or DHT are bulky in nature. Thus, these fluorescent compounds Ij and Ig are useful tools for screening small molecule inhibitors of oxidoreductase or for anti-androgens with small molecules capable of binding to AR.

Although the present disclosure has been described with respect to one or more particular examples, it should be understood that other examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

1. A compound having the structure:

wherein

R₁Is hydrogen or alkyl;

R³selected from the group consisting of carbonyl,

Wherein X is hydroxy and Y is hydrogen or an alkyl group or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or a coumarin isostere group and L³Is optional and is a linking group;

R⁴selected from the group consisting of carbonyl,

Wherein L is¹Is a linking group, L₂Is optional and is a linking group, Z is a terminal group comprising a substituted or unsubstituted carbocyclic group or a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted coumarin isostere group, and R is²Is an alkyl group or hydrogen; and is

R⁵Is a photoactive group which is capable of forming,

with the proviso that when R³Is a carbonyl group or

Or a carbonyl group.

2. The compound of claim 1, wherein L¹And/or L²And/or L³Independently selected from:

wherein n is 1,2,3, 4,5 or 6 and m is 1,2,3, 4,5 or 6.

3. The compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

4. The compound of claim 3, wherein the compound has the structure:

wherein n is 1,2 or 3.

5. The compound of claim 4, wherein the compound has the structure:

6. the compound of claim 1, wherein the compound has the structure:

wherein R is independently selected from L¹-A、R⁵、-OH、-NH₂、-CO₂Et、-CN、-CHO、-SO₃H. and-CO₂H, or three adjacent R groups form a fused ring system, and n is 1,2,3 or 4, and L¹Is a linking group and A is an E3ligase recruitment group wherein only one R is L¹-A。

7. The compound of claim 6, wherein A is formed from:

8. the compound of claim 6, wherein A is formed from a ligase recruitment group that is capable of binding to cerebellum, von Hippel-Linnay (VHL), or mouse double minute 2(MDM 2).

9. The compound of claim 6, wherein the compound has the structure:

10. the compound of claim 6, wherein the compound has the structure:

11. the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

12. The compound of claim 11, wherein the compound has the structure:

wherein n is 1,2 or 3.

13. The compound of claim 12, wherein the compound has the structure:

14. the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

15. The compound of claim 14, wherein the compound has the structure:

wherein n is 1,2 or 3.

16. The compound of claim 15, wherein the compound has the structure:

17. the compound of claim 1, wherein the compound has the structure:

wherein Z is selected from:

and substituted variants thereof, and the use of such substituted variants,

and their substitutionIn a variant of (a) the first and second,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and the use of such substituted variants,

and substituted variants thereof, and

and substituted variants thereof, and the use of such substituted variants,

wherein X' is independently selected at each occurrence from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, and/or a halogen.

18. The compound of claim 17, wherein the compound has the structure:

wherein n is 1,2 or 3.

19. The compound of claim 18, wherein the compound has the structure:

20. the compound of claim 1, wherein the compound has the structure:

wherein R is⁵Selected from:

21. the compound of claim 20, wherein the compound has the structure:

22. a composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

23. The composition of claim 22, further comprising one or more additional drugs.

24. The composition of claim 23, wherein the one or more additional agents are selected from the group consisting of an anti-androgen agent, a 5 α -reductase agent, an androgen metabolism inhibitor agent, and combinations thereof.

25. The composition of claim 24, wherein the anti-androgen drug is selected from the group consisting of flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, dilutamide, and combinations thereof, and/or the 5 a-reductase drug is selected from the group consisting of finasteride, dutasteride, and combinations thereof, and/or the androgen metabolism drug is selected from the group consisting of abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and combinations thereof.

26. The composition of claim 22, wherein the compound has the structure:

or a combination thereof.

27. A method of attenuating Androgen Receptor (AR) transactivation and/or androgen receptor dimerization, comprising:

contacting a cell with the compound of claim 1 in an amount and for a time sufficient to impair androgen receptor transactivation and/or androgen receptor dimerization.

28. The method of claim 27, further comprising contacting a cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, diluxamide, finasteride, dutasteride, or a combination thereof.

29. The method of claim 27, wherein the compound has the structure:

or a combination thereof.

30. A method of inhibiting cell growth comprising contacting a cell with the compound of claim 1 in an amount and for a time sufficient to inhibit cell growth.

31. The method of claim 30, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolumide, dilutamide, finasteride, dutasteride, abiraterone acetate microparticles, ketoconazole, or a combination thereof.

32. The method of claim 30, wherein the compound has the structure:

or a combination thereof.

33. The method of claim 31, wherein the cell growth inhibited is malignant cell growth and/or hyperplastic cell growth.

34. The method of claim 33, wherein the malignant cell growth is a cancer cell growth.

35. The method of claim 34, wherein the cancer cell growth is hormonal cancer cell growth.

36. The method of claim 35, wherein the hormonal cancer is selected from the group consisting of breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof.

37. The method of claim 35, wherein the hormonal cancer cell growth is prostate cancer cell growth.

38. The method of claim 37, wherein the prostate cancer cell growth is castration-resistant prostate cancer cell growth.

39. The method of claim 38, wherein the castration-resistant prostate cancer cells express AR-V7.

40. A method of treating a malignant cell growth disorder and/or a proliferative cell growth disorder in a subject, comprising administering to the subject the compound of claim 1, wherein the malignant cell growth disorder and/or the proliferative cell growth disorder in the subject is treated.

41. The method of claim 40, wherein the malignant cell growth disorder is cancer.

42. The method of claim 41, wherein the cancer is a hormonal cancer.

43. The method of claim 42, wherein the hormonal cancer is selected from the group consisting of breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof.

44. The method of claim 43, wherein the hormonal cancer is prostate cancer.

45. The method of claim 44, wherein the prostate cancer is castration-resistant prostate cancer.

46. The method of claim 45, wherein the cells of castration-resistant prostate cancer express AR-V7.

47. The method of claim 40, wherein the compound has the structure:

or a combination thereof.

48. A method of imaging malignant and/or proliferating cells in an individual, comprising:

administering to the individual one or more compounds of claim 1,

exposing the individual, or a portion thereof, to electromagnetic radiation, thereby exciting one or more compounds,

detecting one or more excited compounds, and

the individual or a part thereof is imaged,

wherein malignant cells and/or hyperproliferative cells are imaged.

49. The method of claim 48, wherein the malignant cell is a cancer cell.

50. The method of claim 49, wherein the cancer cell is a hormonal cancer cell.

51. The method of claim 50, wherein the hormonal cancer cells are selected from the group consisting of breast cancer cells, ovarian cancer cells, uterine or endometrial cancer cells, and prostate cancer cells.

52. The method of claim 51, wherein the hormonal cancer cell is a prostate cancer cell.

53. The method of claim 52, wherein the prostate cancer cell is a castration-resistant prostate cancer cell.

54. The method of claim 53, wherein the castration-resistant prostate cancer cells express AR-V7.

55. A method according to claim 48 wherein imaging is repeated to enable measurement of androgen metabolic activity.

56. The method of claim 48, wherein the compound has the structure:

or a combination thereof.